Method for collecting functional cells in vivo with high efficiency

ABSTRACT

Biologically low invasive vessels are filled with biological factors that have the activity of mobilizing specific functional cells in the body. The vessels are indwelled in the body. After specific functional cells are mobilized into the vessels, the vessels are removed from the body to collect functional cell populations mobilized to the vessels. Alternatively, the cells are directly collected from the vessels indwelled in the body.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of co-pending applicationSer. No. 12/990,086, filed Jan. 4, 2011; which is the National StageApplication of International Application Number PCT/JP2009/058525, filedApr. 30, 2009; which claims priority to Japanese Patent Application No.2008-119355, filed Apr. 30, 2008; which are incorporated herein byreference in their entirety.

The Sequence Listing for this application is labeled“As-filed-ST25.txt”, which was created on Oct. 26, 2010, and is 34 KB.The entire content is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to novel highly efficient and minimallyinvasive methods for collecting functional cells such as stem cells,which exist in an extremely small number in vivo.

BACKGROUND ART

Conventional methods for collecting highly functional cells from theliving body include, for example, methods of collecting hematopoieticbone marrow stem cells, which harvest bone marrow fluid from the bonemarrow of a long bone or pelvic bone by directly inserting a needle intothe bone marrow, and for administration to human, concentrate a stemcell population by centrifugation, and collect and confirm fluorescentlylabeled cells with a cell sorter using stem cell surface markers as anindicator; methods of collecting peripheral blood stem cells, whichmobilize hematopoietic stem cells to peripheral blood by administeringG-CSF, collect peripheral blood and isolate hematopoietic stem cellsfrom the blood; and methods of collecting mesenchymal stem cells, whichisolate mesenchymal stem cells by collecting adherent proliferatingcells from a direct culture of bone marrow fluid, or isolate and culturemesenchymal stem cells from surgically harvested peripheral tissues suchas adipose tissues. However, harvesting bone marrow fluid from the bonemarrow is highly invasive and painful, and involves risk of myelitis dueto intramedullary infection. Thus, the treatment requires highly strictmedical management by experts, and cannot be conducted frequently.Surgical harvest of peripheral tissues also has the same risk. Themobilization of hematopoietic stem cells using G-CSF poses a largeeconomic burden, and also cannot be frequently conducted.

It goes without saying that establishment of efficient and safe methodsfor collecting biologically functional cells will be encouraging newsfor many patients that suffer from intractable diseases and are in needof such cells.

PRIOR ART DOCUMENTS Non-Patent Documents

[Non-patent Document 1] Transplantation of hematopoietic stem cells fromthe peripheral blood. J Cell Mol Med. 2005; 9: 37-50

[Non-patent Document 2] Role of mesenchymal stem cells in regeneratemedicine: application to bone and cartilage repair. Expert Opin BiolTher. 2008; 8: 255-268

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

An objective of the present invention is to provide novel highlyefficient and minimally invasive techniques for collecting functionalcells such as stem cells, which exist in an extremely small number invivo.

Means for Solving the Problems

The present invention provides brand new, highly efficient techniquesfor harvesting biologically functional cells by only implanting a tubeinto a living body in a minimally invasive manner.

Specifically, cylindrical tubes (which has a length of 10 mm and across-sectional area of about 2 mm², and is open on one side and closedon the other side) made of biologically hypoallergenic silicone werefilled with each of HMGB1, hyaluronic acid, phosphate buffer, or such,and then implanted under the dorsal skin of GFP bone marrow-transplantedmice. The tubes were recovered two weeks after implantation, and cellsmobilized and accumulated in the tubes were collected. Some of the cellswere cultured, and others were analyzed for cell surface markers byFACS. The result shows that as compared to tubes filled with phosphatebuffer, significantly more PDGFRα-positive cells were harvested fromtubes filled with an agent other than phosphate buffer, and these cellpopulations contained mesenchymal stem cells with the ability todifferentiate into bone and cartilage.

Based on the above findings, the present invention provides:

[1] a method for collecting a cell population from a vessel removedoutside the body from under the skin;

[2] a method for harvesting a cell population from a vessel removedoutside the body from under the skin, wherein the cell population ismobilized into the vessel by any one of the materials of (a) to (r)described below, or a mixture of any two or more of the materials of (a)to (r) described below:(a) an HMGB1 protein;(b) a cell that secretes an HMGB1 protein;(c) a vector inserted with a DNA encoding an HMGB1 protein;(d) an HMGB2 protein;(e) a cell that secretes an HMGB2 protein;(f) a vector inserted with a DNA encoding an HMGB2 protein;(g) an HMGB3 protein;(h) a cell that secretes an HMGB3 protein; and(i) a vector inserted with a DNA encoding an HMGB3 protein;(j) an S100A8 protein;(k) a cell that secretes an S100A8 protein;(l) a vector inserted with a DNA encoding an S100A8 protein;(m) an S100A9 protein;(n) a cell that secretes an S100A9 protein;(o) a vector inserted with a DNA encoding an S100A9 protein;(p) hyaluronic acid;(q) a cell or tissue extract; and(r) a heparin-binding fraction of cell or tissue extract;[3] a method for collecting a bone marrow cell, which comprises the stepof isolating a bone marrow cell from a cell population collected from avessel implanted under the skin;[4] a method for collecting a bone marrow cell, which comprises the stepof isolating a bone marrow cell from a cell population collected from avessel implanted under the skin, wherein the cell population ismobilized into the vessel by any one of the materials of (a) to (r)described below, or a mixture of any two or more of the materials of (a)to (r) described below:(a) an HMGB1 protein;(b) a cell that secretes an HMGB1 protein;(c) a vector inserted with a DNA encoding an HMGB1 protein;(d) an HMGB2 protein;(e) a cell that secretes an HMGB2 protein;(f) a vector inserted with a DNA encoding an HMGB2 protein;(g) an HMGB3 protein;(h) a cell that secretes an HMGB3 protein; and(i) a vector inserted with a DNA encoding an HMGB3 protein;(j) an S100A8 protein;(k) a cell that secretes an S100A8 protein;(l) a vector inserted with a DNA encoding an S100A8 protein;(m) an S100A9 protein;(n) a cell that secretes an S100A9 protein;(o) a vector inserted with a DNA encoding an S100A9 protein;(p) hyaluronic acid;(q) a cell or tissue extract; and(r) a heparin-binding fraction of cell or tissue extract;[5] the method of [3] or [4], which comprises the step of collecting thecell population from a vessel removed outside the body before the stepof isolating a bone marrow cell from a cell population;[6] the method of [2] or [4], wherein an extract of cell or tissue isproduced by a method comprising the step of immersing a cell or tissuein a solvent;[7] the method of [2] or [4], wherein a heparin-binding fraction of anextract cell or tissue is produced by a method comprising the steps of:(a) immersing a cell or tissue in a solvent;(b) contacting immobilized heparin with an extract prepared in step (a);and(c) eluting a heparin-binding fraction from the immobilized heparin;[8] a cell population collected by the method of any one of [1], [2],[6], and [7];[9] a bone marrow cell isolated by the method of any one of [3] to [7];[10] a tissue-regenerating agent comprising the cell population of [8];[11] a tissue-regenerating agent comprising the bone marrow cell of [9];[12] a method for collecting a cell population, which comprises thesteps of:(I) implanting a vessel under the skin; and(II) collecting a cell population from the vessel;[13] the method of [12], which comprises the step of administering anyone of (a) to (r) or a mixture of any two or more of (a) to (r) to bloodvessel or muscle after step (I):(a) an HMGB1 protein;(b) a cell that secretes an HMGB1 protein;(c) a vector inserted with a DNA encoding an HMGB1 protein;(d) an HMGB2 protein;(e) a cell that secretes an HMGB2 protein;(f) a vector inserted with a DNA encoding an HMGB2 protein;(g) an HMGB3 protein;(h) a cell that secretes an HMGB3 protein;(i) a vector inserted with a DNA encoding an HMGB3 protein;(j) an S100A8 protein;(k) a cell that secretes an S100A8 protein;(l) a vector inserted with a DNA encoding an S100A8 protein;(m) an S100A9 protein;(n) a cell that secretes an S100A9 protein;(o) a vector inserted with a DNA encoding an S100A9 protein;(p) hyaluronic acid;(q) an extract of a cell or tissue; and(r) a heparin-binding fraction of an extract of a cell or tissue;[14] the method of [12], in which the vessel contains any one, or amixture of any two or more of:(a) an HMGB1 protein;(b) a cell that secretes an HMGB1 protein;(c) a vector inserted with a DNA encoding an HMGB1 protein;(d) an HMGB2 protein;(e) a cell that secretes an HMGB2 protein;(f) a vector inserted with a DNA encoding an HMGB2 protein;(g) an HMGB3 protein;(h) a cell that secretes an HMGB3 protein;(i) a vector inserted with a DNA encoding an HMGB3 protein;(j) an S100A8 protein;(k) a cell that secretes an S100A8 protein;(l) a vector inserted with a DNA encoding an S100A8 protein;(m) an S100A9 protein;(n) a cell that secretes an S100A9 protein;(o) a vector inserted with a DNA encoding an S100A9 protein;(p) hyaluronic acid;(q) an extract of a cell or tissue; and(r) a heparin-binding fraction of a extract of a cell or tissue;[15] a method for collecting a bone marrow cell, which comprises thesteps of:(I) implanting a vessel under the skin;(II) harvesting a cell population from the vessel; and(III) isolating a bone marrow cell from the harvested cell population;[16] the method of [15], which comprises the step of administering anyone of (a) to (r) or a mixture of any two or more of (a) to (r) to bloodvessel or muscle after step (I):(a) an HMGB1 protein;(b) a cell that secretes an HMGB1 protein;(c) a vector inserted with a DNA encoding an HMGB1 protein;(d) an HMGB2 protein;(e) a cell that secretes an HMGB2 protein;(f) a vector inserted with a DNA encoding an HMGB2 protein;(g) an HMGB3 protein;(h) a cell that secretes an HMGB3 protein;(i) a vector inserted with a DNA encoding an HMGB3 protein;(j) an S100A8 protein;(k) a cell that secretes an S100A8 protein;(l) a vector inserted with a DNA encoding an S100A8 protein;(m) an S100A9 protein;(n) a cell that secretes an S100A9 protein;(o) a vector inserted with a DNA encoding an S100A9 protein;(p) hyaluronic acid;(q) a cell or tissue extract; and(r) a heparin-binding fraction of cell or tissue extract;[17] the method of [15], in which the vessel contains any one, or amixture of any two or more of:(a) an HMGB1 protein;(b) a cell that secretes an HMGB1 protein;(c) a vector inserted with a DNA encoding an HMGB1 protein;(d) an HMGB2 protein;(e) a cell that secretes an HMGB2 protein;(f) a vector inserted with a DNA encoding an HMGB2 protein;(g) an HMGB3 protein;(h) a cell that secretes an HMGB3 protein;(i) a vector inserted with a DNA encoding an HMGB3 protein;(j) an S100A8 protein;(k) a cell that secretes an S100A8 protein;(l) a vector inserted with a DNA encoding an S100A8 protein;(m) an S100A9 protein;(n) a cell that secretes an S100A9 protein;(o) a vector inserted with a DNA encoding an S100A9 protein;(p) hyaluronic acid;(q) an extract of a cell or tissue; and(r) a heparin-binding fraction of cell or tissue extract;[18] the method of any one of [13], [14], [16], and [17], in which theextract of a cell or tissue is produced by a method comprising the stepof immersing a cell or tissue in a solvent;[19] the method of any one of [13], [14], [16], and [17], in which theheparin-binding fraction of the extract of a cell or tissue is producedby a method comprising the steps of:(a) immersing a cell or tissue in a solvent;(b) contacting immobilized heparin with an extract prepared in step (a);and(c) eluting a heparin-binding fraction from the immobilized heparin;[20] a cell population collected by the method of any one of [12] to[14];[21] a bone marrow cell isolated by the method of any one of [15] to[17];[22] a tissue-regenerating agent comprising the cell population of [20];and[23] a tissue-regenerating agent comprising the bone marrow cell of[21].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows in a schematic diagram a high-efficiency method forcollecting biologically functional cells. A hypoallergenic tube(silicone tube or such) filled with a factor that specifically mobilizesbiologically functional cells is placed in the body. As a result,functional cells are selectively mobilized into the tube from theperipheral blood circulation or tissue.

FIG. 2 shows in a photograph that cells accumulated in a tube(tube-entrapping cells; TECs) are GFP-positive.

FIGS. 3A-3B show in a set of photographs TECs 24 hours after start ofculture. FIG. 3A shows a light-field image of proliferatingfibroblast-like cells and epithelial cell-like cells adhering to aplastic culture dish. FIG. 3B shows a GFP fluorescence image of the darkfield.

FIGS. 4A-4B show in a set of photographs assessment of TECs collectedfrom a tube for their ability to differentiate into osteoblasts. Cellsharvested from the tube were cultured in an osteoblastdifferentiation-inducing culture medium and were confirmed todifferentiate into osteoblasts positive for alizarin red stain in abouttwo weeks.

FIGS. 5A-5B show in a set of photographs assessment of TECs collectedfrom a tube for their ability to differentiate into adipocytes. Cellsharvested from the tube were cultured in an adipocytedifferentiation-inducing culture medium and were confirmed todifferentiate into adipocytes positive for oil red stain in about twoweeks.

FIG. 6 shows in a set of photographs assessment of TECs collected from atube for their ability to differentiate into epidermal cells. Cellscollected from the tube were cultured in an epidermal celldifferentiation-inducing culture medium and were confirmed todifferentiate into epidermal cells expressing keratinocyte-specifickeratin 5 in about two weeks.

FIG. 7 shows in a set of diagrams assessment of the expression of PDGFRαand CD44 on TECs. Collection of PDGFRα and CD44 double-positive cells inthe tube was confirmed.

FIG. 8 shows in a set of photographs Western blot detection of the HMGBfamily in neonatal mouse skin extract.

FIG. 9 shows in a diagram an HMGB1 expression vector.

FIG. 10 shows in a set of photographs Western blot results for thepurified recombinant Flag tag-HMGB family-fusion proteins expressed inHEK293 cells.

FIGS. 11A-11C show in a set of graphs the chemotactic activity ofrecombinant HMGB1/HMGB2/HMGB3 for bone marrow mesenchymal stem cellsusing a Boyden chamber. All recombinant proteins showed higherchemotactic activities as compared to the control groups.

FIGS. 12A-12C show in a set of graphs the result of treatment in a mousecutaneous ulcer treatment model using the HMGB family proteins. HMGB1,HMGB2, and HMGB3 all showed significant effects on reducing the ulcerarea as compared to the control groups.

FIG. 13 shows in a photograph the activity of human HMGB1 and a humanskin extract to induce the migration of human bone marrow-derivedmesenchymal stem cells as confirmed using a Boyden chamber.

FIG. 14 shows in a set of photographs the activity of activatorspurified on a heparin column from mouse heart, brain, and skin extractsto induce bone marrow mesenchymal stem cells as confirmed using a Boydenchamber.

FIGS. 15A-15B show in a set of photographs the activity of an extract ofthe cultured cell line HEK293 or HeLa to induce migration of human bonemarrow mesenchymal stem cells as confirmed using a Boyden chamber. Bothcultured cell lines showed chemotactic activity for human bone marrowmesenchymal stem cells.

FIG. 16A shows in a photograph a mouse fixed to a brain stereotaxicapparatus and subjected to a midline incision in the head with ascalpel, followed by trepanation using a drill. FIG. 16B shows in aphotograph a brain to which a negative pressure is applied using asyringe to suck part of the brain tissue. FIG. 16C is a photograph of amouse after injection of 5 μl heparin-column purified fraction of a skinextract dissolved in fibrin adhesive formulation (fibrinogen), and asubsequent injection of 5 μl of fibrin glue formulation (thrombin).FIGS. 16D and 16E are photographs of the brain injury model taken 2weeks after the treatment. Higher accumulation of GFP-positive cells wasobserved in the treatment group using the heparin-column purifiedfraction of skin extract in E compared to the control in D. FIGS. 16Fand 16G are photographs of the brain injury model taken 6 weeks afterthe treatment. Higher accumulation of GFP-positive cells was observed inthe treatment group using the heparin-column purified fraction of skinextract in G compared to the control in F.

FIG. 17 shows in a photograph assay results of measuring the migratoryactivity of bone-marrow derived mesenchymal stem cells in skin extractsusing a Boyden chamber. These images show blue-stained bone marrowmesenchymal stem cells, which have migrated from the upper compartmentof the Boyden chamber through a 8-μm micropore polycarbonate membranefilter into the lower compartment containing skin extracts, and adheredto the lower-compartment side of the membrane. Skin extracts collectedfrom two-day-old or six-week-old mice were placed in the lower chambers.

FIGS. 18A-18B show in a set of photographs Western blot detection of theS100A8 (FIG. 18A) and S100A9 (FIG. 18B) proteins in skin extracts.

FIG. 19 shows in a photograph elution of a heparin-binding protein inskin extracts eluted from a heparin affinity column by a concentrationgradient of NaCl. Proteins in each fraction were separated by SDS-PAGEand detected by silver staining.

FIG. 20 shows in a photograph assay results of measuring the migratoryactivity of bone marrow-derived mesenchymal stem cells in skin extractsusing a Boyden chamber. The image shows blue-stained bone marrowmesenchymal stem cells, which have migrated from the upper compartmentof the Boyden chamber through the micropores of a filter to eachheparin-binding fraction in skin extracts (to the lower compartment),and adhered to the lower-compartment side of the membrane.

FIGS. 21A-21B show in a set of photographs Western blot detection of theS100A8 (FIG. 21A) and S100A9 (FIG. 21B) proteins in each heparin-bindingfraction of skin extracts.

FIG. 22 shows in a diagram the expression vector for S100A8 or S100A9.

FIG. 23 shows in a photograph assay results of measuring the migratoryactivity of bone marrow-derived mesenchymal stem cells in skin extractsusing a Boyden chamber. These images show blue-stained bone marrowmesenchymal stem cells, which have migrated from the upper compartmentof the Boyden chamber through the micropores of a filter into the lowercompartment containing recombinant GST-S100A8, GST-S100A9, or skinextracts, and adhered to the lower-compartment side of the membrane.

FIG. 24A shows in a set of diagrams the FACS results for CD44, PDGFRα,and PDGFRβ in the CD45-negative cell fraction in peripheral blood 12hours after administration of GST-S100A8 or GST-S100A via the mousecaudal vein. FIG. 24B shows in graphs quantitative analysis of thepopulations of CD45-negative, CD44-positive, PDGFRα-positive cells, orCD45-negative, CD44-positive, PDGFRβ-positive cells in peripheral blood12 hours after administration of GST-S100A8 or GST-S100A based on theFACS results.

FIG. 25 shows in a graph therapeutic effect of S100A8 on cutaneous ulcerin normal mice.

FIG. 26 shows in a graph therapeutic effect of S100A8 on cutaneous ulcerin diabetic mice.

FIG. 27 shows in a graph therapeutic effect of cells that are mobilizedinto a device using skin extracts or peripheral blood extracts oncutaneous ulcer.

FIGS. 28A-28D show in a set of photographs fluorescence microscopicdetection of bone marrow cells mobilized into a device using heparinaffinity column-binding components of peripheral blood extracts.

FIG. 29 shows in a graph detection of bone marrow cells mobilized into adevice using heparin affinity column-binding components of peripheralblood extracts by fluorescence microscopy, and quantification of thenumber of bone marrow cells using an image analysis software.

FIGS. 30A-30E show in a graph detection of bone marrow-derived cells(GFP-positive cells) mobilized into a device using S100A8, HMGB1, HMGB2,or HMGB3 (FIG. 30A, S100A8; FIG. 30B, HMGB1; FIG. 30C, HMGB2; FIG. 30D,HMGB3; FIG. 30E, negative control) by fluorescence microscopy.

FIG. 31 shows in a set of photographs therapeutic effect on cutaneousulcer developed in BALB/cAJcl-nu/nu mice, of bone marrow-derived cellsmobilized into a device using S100A8, HMGB1, or HMGB2.

FIG. 32 shows in a diagram an HMGB1 expression vector.

FIG. 33 shows in a diagram administration of skin extract (SE) to amouse via caudal vein, followed by collection of peripheral blood.

FIG. 34 shows in a diagram flow cytometric fractionation of a mouseperipheral blood mononuclear cell fraction fluorescently labeled withanti-mouse PDGFRα antibody and anti-mouse CD44 antibody 12 hours afteradministration of skin extract (SE). The upper three charts correspondto the PBS-administered group (n=3) as a negative control, while thelower three charts correspond to the skin extract (SE)-administeredgroup (n=3). The vertical axis indicates the expression level of CD44,and the horizontal axis indicates the expression level of PDGFRα. Thearea boxed with blue line corresponds to a population of CD44 and PDGFRαdouble-positive cells. The population was increased in the skinextract-administered group (SE) as compared to the PBS-administeredgroup.

FIG. 35 shows in a diagram administration of HMGB1 to a mouse via caudalvein, followed by collection of peripheral blood.

FIG. 36 shows in a diagram flow cytometric fractionation of mouseperipheral blood mononuclear cell fraction fluorescently labeled withanti-mouse PDGFRα antibody and anti-mouse CD44 antibody 12 hours afteradministration of HMGB1. The left chart corresponds to thePBS-administered mice as a negative control, while the right chartcorresponds to the HMGB1-administered mice. The vertical axis indicatesthe expression level of CD44, and the horizontal axis indicates theexpression level of PDGFRα. The area boxed with blue line corresponds toa population of CD44 and PDGFRα double-positive cells. The populationwas increased in the HMGB1-administered mice as compared to thePBS-administered mice.

FIG. 37A shows in a diagram the flow cytometry result that shows thepresence of cells having CD44 and PDGFRα. HMGB1 administration increasedboth populations of PDGFRα and CD44 double-positive cells, andPDGFRα-positive CD44-negative cells in peripheral blood. FIGS. 37B and37C show results of comparison between the PBS- and HMGB1-administeredgroups on the presence of PDGFRα and CD44 double-positive cells, andPDGFRα-positive CD44-negative cells in peripheral blood, respectively.Both cell populations were statistically significantly increased in theHMGB1-administered group.

MODE FOR CARRYING OUT THE INVENTION

The present invention provides a method for collecting a cell populationfrom a vessel removed outside the body from under the skin.

The present invention also provides a method for collecting a bonemarrow cell, which comprises the step of isolating a bone marrow cellfrom a cell population collected from a vessel implanted under the skin.The above method may include the step of collecting the cell populationfrom the vessel removed outside the body before the step of isolating abone marrow cell from a cell population.

The present invention provides a method for collecting a cellpopulation, which comprises the steps of:

(I) implanting a vessel under the skin; and

(II) collecting a cell population from the vessel.

Furthermore, the present invention provides a method for collecting abone marrow cell, which comprises the steps of:

(I) implanting a vessel under the skin;

(II) harvesting a cell population from the vessel; and

(III) isolating a bone marrow cell from the harvested cell population.

Alternatively, the methods described above may comprise, subsequent tostep (I), the step of removing the vessel from under the skin.

Preferred materials for the above-described vessel include, but are notlimited to, silicone, vinyl, plastic, and other biologicallyhypoallergenic materials. Meanwhile, the size of vessel used in theExamples was: 10 mm length×2 mm cross-sectional area (with a volume of20 ml; for mice); however, the size is not limited to the above example,as long as the vessel can be implanted under the skin. The wallthickness of vessel used in the Examples was about 0.5 mm; however, thethickness is not limited to the above examples, as long as it issufficient to maintain adequate strength. The shape of vessel used inthe Examples was a cylindrical shape that is open only on one side;however, the shape is not particularly limited, so long as it does notdamage biological tissues; and the shape includes cylindrical, spindle,spherical, and ovoid. Vessels used in the present invention includesilicone tubes, vinyl bags, and indwelling injection needles. Thevessels are not particularly limited, as long as they are implantablemedical materials or devices in vivo.

In the present invention, cell populations harvested from the vesselinclude bone marrow-derived cells.

Bone marrow cells of the present invention are cells other thanhematopoietic stem cells, or cells derived therefrom such as leukocytes,erythrocytes, and platelets, and include stem cells represented by cellswhich have been hitherto called bone marrow mesenchymal stem cells, bonemarrow stromal pluripotent stem cells, or bone marrow pluripotent stemcells and tissue progenitor cell populations existing in the bonemarrow. Bone marrow cells of the present invention can be isolated frombone-marrow collection (bone marrow cell collection) or peripheral bloodcollection. Hematopoietic stem cells are nonadherent, while bone marrowcells of the present invention are obtained as adherent cells by meansof a cell culture of a mononuclear cell fraction of blood obtained fromthe bone marrow collection (bone marrow cell collection) or peripheralblood collection. Moreover, bone marrow cells of the present inventioninclude mesenchymal stem cells, and have a potential to differentiateinto, preferably, osteoblasts (the induction of differentiation can beidentified by observing calcification), chondrocytes (which can beidentified by alcian blue positive staining, safranin O positivestaining, or the like), adipocytes (which can be identified by Sudan IIIpositive staining), and other mesenchymal cells such as fibroblasts,smooth muscle cells, stromal cells, and tendon cells; and further nervecells, epithelial cells (for example, epidermal keratinocytes andintestinal epithelial cells express cytokeratin family), and vascularendothelial cells. However, the cells to be differentiated into are notlimited to the above cells, and the potential to differentiate intocells of parenchymatous organs such as liver, kidney, and pancreas arealso included.

In the present invention, bone marrow-derived mesenchymal stem cells,bone marrow stromal pluripotent stem cells, or bone marrow pluripotentstem cells refer to cells existing in the bone marrow, which aredirectly collected from the bone marrow or indirectly collected fromother tissues (blood, skin, adipose, and other tissues), and can becultured/proliferated as adherent cells on a culture dish (made ofplastic or glass). These cells are characterized in having a potentialto differentiate into mesenchymal tissues (mesenchymal stem cells) suchas bone, cartilage, and adipose, or skeletal muscles, heart muscles,further, nerve tissues, epithelial tissues (pluripotent stem cells) andcan be obtained from a collection of bone marrow blood, peripheralblood, or mesenchymal tissues such as adipose, epithelial tissues suchas skin, nerve tissues such as brain. Bone marrow-derived mesenchymalstem cells, bone marrow-derived pluripotent stem cells, or bone marrowpluripotent stem cells are also characterized in having a potential todifferentiate into epithelial tissues such as keratinocytes thatconstitute skin or into nerve tissues that constitute brain, byadministrating these cells that have once adhered onto a culture dish toa lesion area of the living body.

Bone marrow mesenchymal stem cells, bone marrow stromal pluripotent stemcells, or bone marrow pluripotent stem cells of the present inventionare multipotent stem cells, and have a potency to differentiatepreferably into: osteoblasts (the induction of differentiation can beidentified by observing calcification), chondrocytes (which can beidentified by alcian blue positive staining, safranin O positivestaining, or the like), adipocytes (which can be identified by Sudan IIIpositive staining or the like), and other mesenchymal cells such asfibroblasts, smooth muscle cells, skeletal muscle cells, stromal cells,and tendon cells; nerve cells, pigment cells, epidermal cells, hairfollicle cells (which express cytokeratin family, hair keratin family,or the like), epithelial cells (for example, epidermal keratinocytes andintestinal epithelial cells express cytokeratin family or the like), andendothelial cells; and further preferably into cells of parenchymatousorgans such as liver, kidney, and pancreas. However, differentiatedcells are not limited to the above cells.

Tissue progenitor cells are defined as undifferentiated cells having aunidirectional potency to differentiate into specific tissue cells otherthan the blood system, and include undifferentiated cells having thepotency to differentiate into mesenchymal tissue, epithelial tissue,nerve tissue, parenchymatous organs, and vascular endothelium asmentioned above.

Meanwhile, bone marrow cells of the present invention include, but arenot limited to, for example, bone marrow cells positive for at least oneof the cell surface markers: CD44, PDGFRα, and PDGFRβ.

In the present invention, the step of implanting a vessel under the skinis achieved by performing skin incision of several millimeters with ascalpel after general (or local) anesthesia; creating a necessary spacein subcutaneous adipose tissue by blunt dissection using a round-endedmetal rod (mosquito forceps or such); implanting a cell harvestingvessel such as a silicone tube into the space; and finally closing theincision by suture or stapling.

Possible alternative methods for indwelling vessels under the skininclude methods which insert the inner and outer sheaths of an injectionneedle under the skin, and then removing the inner sheath (injectionneedle) and leaving the outer sheath in; and methods which insertballoon catheter over a guide wire under the skin, and indwelling thecatheter after removing the guide wire by expanding its balloon with apharmaceutical fluid.

Subjects of the vessel implantation include humans and nonhuman animals,including, for example, humans, mice, rats, monkeys, pigs, dogs,rabbits, hamsters, and guinea pigs. The preferred subject is humans.

In the present invention, the step of collecting cell populations fromvessels may be either the step of harvesting cell populations fromvessels implanted under the skin, or the step of removing the vesselsfrom under the skin, followed by collecting cell populations from theremoved vessels. For example, the Examples herein describe that siliconetubes implanted under the skin were recovered via skin incision andcells were collected from the tubes by aspiration using pipettes.However, other methods are also available.

Methods for collecting cell populations from vessels implanted under theskin are carried out as follows: a vessel to be implanted under the skinis modified to be elongated to form a tube-shaped end; the tube-likeportion is fixed outside the body; a syringe is attached to the tube atthe time of cell harvest; and the cells are harvested by vacuumaspiration. Then, a cell inducing solution such as HMGB1 solution isre-injected into the tube implanted in the body. This enables repeatedcell harvest from the vessel implanted under the skin. Vessels whoseshape is suitable for the methods are implanted in the body.

In the present invention, the step of isolating bone marrow cells fromthe harvested cell populations is achieved, for example, by isolatingadherent cells adhered to culture dishes from the harvested cellpopulations.

Another method, for example, harvests cells by pipetting from tubesremoved from the body; reacting at least one of the cell surfacemarkers, CD44, PDGFRα, and PDGFRβ using antibodies labeled withdifferent types of fluorescent labels; and then using a cell sorter toisolate cell populations having specific cell surface markers with thepresence of each type of fluorescence as an indicator.

There is an alternative method (MACS), which is performed, for example,as follows: cell surface markers are reacted in the same way withantibodies labeled with metal particles instead of fluorescence; cellsbound with the metal-linked antibodies are attracted and immobilizedonto the internal wall of one side of the tube using magnetic force;after thoroughly eluting cells nonreactive to the antibodies, targetcells immobilized in the tube are collected by releasing the magneticforce.

The present invention provides methods for collecting cell populationsfrom vessels removed to the outside of the body from under the skin, inwhich any one of the materials of (a) to (r), or a mixture of any two ormore of the materials of (a) to (r) described below are responsible formobilizing the cell populations into the vessels from the bone marrow.

The present invention also provides methods for collecting bone marrowcells, which comprise the step of isolating bone marrow cells from cellpopulations harvested from vessels implanted under the skin, and inwhich any one of the materials of (a) to (r) described below, or amixture of any two or more of the materials of (a) to (r) describedbelow are responsible for mobilizing the cell populations to the vesselsfrom the bone marrow. The above methods may comprise the step ofcollecting cell populations from vessels removed from the body beforethe step of isolating bone marrow cells from the cell populations.

(a) an HMGB1 protein;

(b) a cell that secretes an HMGB1 protein;

(c) a vector inserted with a DNA encoding an HMGB1 protein;

(d) an HMGB2 protein;

(e) a cell that secretes an HMGB2 protein;

(f) a vector inserted with a DNA encoding an HMGB2 protein;

(g) an HMGB3 protein;

(h) a cell that secretes an HMGB3 protein; and

(i) a vector inserted with a DNA encoding an HMGB3 protein;

(j) an S100A8 protein;

(k) a cell that secretes an S100A8 protein;

(I) a vector inserted with a DNA encoding an S100A8 protein;

(m) an S100A9 protein;

(n) a cell that secretes an S100A9 protein;

(o) a vector inserted with a DNA encoding an S100A9 protein;

(p) hyaluronic acid;

(q) an extract of cells or tissues; and

(r) a heparin-binding fraction of cell or tissue extract.

The present invention provides methods comprising the steps of:

(I) implanting a vessel under the skin; and

(II) harvesting a cell population from the vessel;

and further comprising subsequent to step (I), the step of administeringto blood vessel or muscle any one, or a mixture of any two or more ofthe materials of (a) to (r) described above.

To achieve step (II), a cell population may be collected from a vesselin the body or a vessel removed from the body.

The present invention also provides methods for collecting bone marrowcells, which comprise the steps of:

(I) implanting a vessel under the skin;

(II) collecting a population of cells from the vessel; and

(III) isolating bone marrow cells from the harvested cell population;

and further comprising subsequent to step (I), the step of administeringto blood vessel or muscle any one of the materials of (a) to (r)described above, or a mixture of any two or more of the materials of (a)to (r) described above.

To achieve step (II), a cell population may be harvested from a vesselin the body or a vessel removed from the body.

The present invention provides methods for collecting cell populations,which comprise the steps of:

(I) implanting a vessel under the skin; and

(II) collecting a population of cells from the vessel;

where the vessel contains any one of the materials of (a) to (r)described above, or a mixture of any two or more of the materials of (a)to (r) described above.

To achieve step (II), cell populations may be harvested from a vesselimplanted in the body or a vessel removed from the body.

The present invention also provides methods for collecting bone marrowcells, which comprise the steps of:

(I) implanting a vessel under the skin;

(II) collecting a population of cells from the vessel; and

(III) isolating bone marrow cells from the harvested cell population;

where the vessel contains any one of the materials of (a) to (r)described above, or a mixture of any two or more of the materials of (a)to (r) described above.

To achieve step (II), a cell population may be collected from a vesselimplanted in the body or a vessel removed from the body.

The present invention also provides methods for collecting cellpopulations, which comprise the steps of:

(I) implanting a vessel under the skin; and

(II) collecting a population of cells from the vessel:

where the vessel contains any one of the materials of (a) to (r)described above, or a mixture of any two or more of the materials of (a)to (r) described above;

and which also comprise subsequent to step (I), the step ofadministering to blood vessel or muscle, any one of the materials of (a)to (r) described above, or a mixture of any two or more of the materialsof (a) to (r) described above.

To achieve step (II), a cell population may be collected from a vesselimplanted in the body or a vessel removed from the body.

The present invention also provides methods for collecting bone marrowcells, which comprise the steps of:

(I) implanting a vessel under the skin;

(II) collecting a population of cells from the vessel; and

(III) isolating bone marrow cells from the harvested cell population;

where the vessel contains any one of the materials of (a) to (r)described above, or a mixture of any two or more of the materials of (a)to (r) described above;

and which also comprise subsequent to step (I), the step ofadministering to blood vessel or muscle any one of the materials of (a)to (r) described above, or a mixture of any two or more of the materialsof (a) to (r) described above.

To achieve step (II), a cell population may be harvested from a vesselimplanted in the body or a vessel removed from the body.

The HMGB1 protein of the present invention can be exemplified by, but isnot limited to proteins comprising the amino acid sequence of SEQ ID NO:1, 3, or 5. HMGB1 proteins of the present invention can also includeproteins which are functionally equivalent to the protein comprising theamino acid sequence of SEQ ID NO: 1, 3, or 5. Examples of such proteinsinclude: 1) isolated proteins which comprise an amino acid sequence withone or more amino acid substitutions, deletions, insertions, and/oradditions in the amino acid sequence of SEQ ID NO: 1, 3, or 5, and whichare functionally equivalent to the protein comprising the amino acidsequence of SEQ ID NO: 1, 3, or 5; and 2) isolated proteins which areencoded by DNAs that hybridize under stringent conditions with DNAscomprising the nucleotide sequence of SEQ ID NO: 2, 4, or 6, and whichare functionally equivalent to the protein comprising the amino acidsequence of SEQ ID NO: 1, 3, or 5.

The HMGB2 protein of the present invention can be exemplified by, but isnot limited to proteins comprising the amino acid sequence of SEQ ID NO:7, 9, or 11. HMGB2 proteins of the present invention can also includeproteins which are functionally equivalent to the protein comprising theamino acid sequence of SEQ ID NO: 7, 9, or 11. Examples of such proteinsinclude: 1) isolated proteins which comprise an amino acid sequence withone or more amino acid substitutions, deletions, insertions, and/oradditions in the amino acid sequence of SEQ ID NO: 7, 9, or 11, andwhich are functionally equivalent to the protein comprising the aminoacid sequence of SEQ ID NO: 7, 9, or 11; and 2) isolated proteins whichare encoded by DNAs that hybridize under stringent conditions with DNAscomprising the nucleotide sequence of SEQ ID NO: 8, 10, or 12, and whichare functionally equivalent to the protein comprising the amino acidsequence of SEQ ID NO: 7, 9, or 11.

The HMGB3 protein of the present invention can be exemplified by, but isnot limited to proteins comprising the amino acid sequence of SEQ ID NO:13 or 15. HMGB3 proteins of the present invention can also includeproteins which are functionally equivalent to the protein comprising theamino acid sequence of SEQ ID NO: 13 or 15. Examples of such proteinsinclude: 1) isolated proteins which comprise an amino acid sequence withone or more amino acid substitutions, deletions, insertions, and/oradditions in the amino acid sequence of SEQ ID NO: 13 or 15, and whichare functionally equivalent to the protein comprising the amino acidsequence of SEQ ID NO: 13 or 15; and 2) isolated proteins which areencoded by DNAs that hybridize under stringent conditions with DNAscomprising the nucleotide sequence of SEQ ID NO: 14 or 16, and which arefunctionally equivalent to the protein comprising the amino acidsequence of SEQ ID NO: 13 or 15.

The S100A8 protein of the present invention can be exemplified by, butis not limited to proteins comprising the amino acid sequence of SEQ IDNO: 17, 19, or 21. S100A8 proteins of the present invention can alsoinclude proteins which are functionally equivalent to the proteincomprising the amino acid sequence of SEQ ID NO: 17, 19, or 21. Examplesof such proteins include: 1) isolated proteins which comprise an aminoacid sequence with one or more amino acid substitutions, deletions,insertions, and/or additions in the amino acid sequence of SEQ ID NO:17, 19, or 21, and which are functionally equivalent to the proteincomprising the amino acid sequence of SEQ ID NO: 17, 19, or 21; and 2)isolated proteins which are encoded by DNAs that hybridize understringent conditions with DNAs comprising the nucleotide sequence of SEQID NO: 18, 20, or 22, and which are functionally equivalent to theprotein comprising the amino acid sequence of SEQ ID NO: 17, 19, or 21.

The S100A9 protein of the present invention can be exemplified by, butis not limited to, proteins comprising the amino acid sequence of SEQ IDNO: 23, 25, or 27. S100A9 proteins of the present invention can alsoinclude proteins which are functionally equivalent to the proteincomprising the amino acid sequence of SEQ ID NO: 23, 25, or 27. Examplesof such proteins include: 1) isolated proteins which comprise an aminoacid sequence with one or more amino acid substitutions, deletions,insertions, and/or additions in the amino acid sequence of SEQ ID NO:23, 25, or 27, and which are functionally equivalent to the proteincomprising the amino acid sequence of SEQ ID NO: 23, 25, or 27; and 2)isolated proteins which are encoded by DNAs that hybridize understringent conditions with DNAs comprising the nucleotide sequence of SEQID NO: 24, 26, or 28, and which are functionally equivalent to theprotein comprising the amino acid sequence of SEQ ID NO: 23, 25, or 27.

Isolated proteins which are functionally equivalent to the proteincomprising the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, or 27 may be homologues or paralogues to theprotein comprising the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, or 27. Those skilled in the art canisolate proteins which are functionally equivalent to the proteincomprising the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, or 27, by known methods (supplementary volume of“Jikken Igaku (Experimental Medicine), Idenshi Kougaku Handbook (GeneticEngineering Handbook)”, pp 246-251, published by Yodosha Co., Ltd.,1991).

Examples of proteins which are functionally equivalent to the proteincomprising the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, or 27 include proteins having bonemarrow-derived cell-inducing activity.

Proteins which comprise an amino acid sequence with one or more aminoacid substitutions, deletions, insertions, and/or additions in the aminoacid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, or 27, and which are functionally equivalent to the proteincomprising the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, or 27 include naturally-occurring proteins.Generally, eukaryotic genes have polymorphism as known in interferongenes and such. Alterations in nucleotide sequence caused by thepolymorphism may result in one or more amino acid substitutions,deletions, insertions, and/or additions. Naturally-occurring proteinssuch as those comprising an amino acid sequence with one or more aminoacid substitutions, deletions, insertions, and/or additions in the aminoacid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, or 27, and which are functionally equivalent to the proteincomprising the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, or 27 are included in HMGB1, HMGB2, HMGB3,S100A8, or S100A9 proteins of the present invention.

The present invention also includes artificially-produced mutantproteins as long as they are functionally equivalent to the proteincomprising the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, or 27. Known methods which cause randommutations to a given nucleotide sequence include substitution(s) of basepair(s) through nitrous acid treatment of DNA (Hirose, S. et al., Proc.Natl. Acad. Sci. USA., 79: 7258-7260, 1982). This method enables randomintroduction of substitution(s) of base pair(s) into a specific segmentby nitrous acid treatment of the segment desired to be mutated.Alternatively, technologies for site-directing a target mutation includethe gapped duplex method (Kramer W. and Fritz H J., Methods in Enzymol.,154: 350-367, 1987) and the like. A cyclic double stranded vector inwhich a gene to be introduced with a mutation is cloned, is separatedinto single strands. These single strands are hybridized with asynthetic oligonucleotide mutated at the target site. A vector-derivedcomplementary single strand DNA linearized by a restriction enzyme isannealed with the cyclic single stranded vector, and the gap between theoligonucleotide and the vector is filled by using a DNA polymerase,which is then made into a complete double stranded vector by ligation.

The number of amino acids to be modified would be typically within 50,preferably within 30, and more preferably within 5 amino acids (forexample, one amino acid).

When an amino acid is artificially substituted, substitution with anamino acid having similar properties would result in maintaining theactivity of the original protein. Proteins of the present inventioninclude proteins resulting from a conservative substitution in the abovesubstitution of amino acid(s), and which are functionally equivalent tothe protein comprising the amino acid sequence of SEQ ID NO: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. Conservative substitution isconsidered important when substituting amino acid(s) of domainsimportant for protein activities. Such a conservative substitution ofamino acid(s) is well known to those skilled in the art.

Examples of amino acid groups suitable for conservative substitutioninclude basic amino acids (such as lysine, arginine, and histidine),acidic amino acids (such as aspartic acid and glutamic acid), unchargedpolar amino acids (such as glycine, asparagine, glutamine, serine,threonine, tyrosine, and cysteine), nonpolar amino acids (such asalanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, and tryptophane), β branched amino acids (such as threonine,valine, and isoleucine), and aromatic amino acids (such as tyrosine,phenylalanine, tryptophane, and histidine).

Moreover, non-conservative substitution may increase protein activities(for example, constitutively activated proteins).

In addition, proteins which are functionally equivalent to the proteincomprising the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, or 27 can be obtained by methods that utilizehybridization. That is to say, a DNA encoding HMGB1, HMGB2, HMGB3,S100A8, or S100A9 protein of the present invention as shown in the SEQID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28 or afragment thereof is used as a probe, and then DNAs that can hybridize tothem are isolated. A hybridization reaction performed under stringentconditions leads to the selection of highly homologous DNA as anucleotide sequence. This increases the chances of isolated proteinscontaining proteins that are functionally equivalent to the HMGB1,HMGB2, HMGB3, S100A8, or S100A9 protein. Examples of a highly homologousnucleotide sequence include those having 70% or more, and desirably 90%or more identity.

In a specific example, the term “stringent conditions” refers tohybridization conditions with 6×SSC, 40% formamide at 25° C. andsubsequent washing with 1×SSC at 55° C. The stringency depends onconditions such as salt concentration, formamide concentration, ortemperature; however it is obvious for those skilled in the art to setthese conditions so as to obtain necessary stringency.

With the use of hybridization, for example, DNAs encoding homologues ofthe HMGB1, HMGB2, HMGB3, S100A8, or S100A9 proteins other than thoseproteins comprising the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, or 27 can be isolated.

Proteins which are functionally equivalent to a protein comprising theamino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, or 27 normally have a high homology with the amino acid sequenceof SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. Theterm “high homology” refers to a sequence identity of at least 30% ormore, preferably 50% or more, more preferably 80% or more (for example,95% or more). The identity of the nucleotide sequences and amino acidsequences can be determined using a homology search site via theinternet (For example, homology searches such as FASTA, BLAST,PSI-BLAST, and SSEARCH can be used in the DNA Data Bank of Japan (DDBJ)[examples of which include the homology search page (Search andAnalysis) at the DNA Data Bank of Japan (DDBJ) website;http://www.ddbj.nig.ac.jp/E-mail/homology-j.html]). Furthermore,searches using BLAST can be carried out through the web site of theNational Center for Biotechnology Information (NCBI) (examples of whichinclude BLAST page at the homepage of NCBI website;http://www.ncbi.nlm.nih.gov/BLAST/; Altschul, S. F. et al., J. Mol.Biol., 1990, 215(3): 403-10; Altschul, S. F. & Gish, W., Meth. Enzymol.,1996, 266: 460-480; Altschul, S. F. et al., Nucleic Acids Res., 1997,25: 3389-3402)).

For example, in the calculation of the identity of amino acid sequencesusing Advanced BLAST 2.1, the identity value (%) can be obtained by thefollowing: blastp is used as the program, expect value is set at 10, allfilters are set at OFF, BLOSUM62 is used for matrix, and gap existencecost, per residue gap cost, and lambda ratio are set at 11, 1, and 0.85,respectively (default parameters) (Karlin, S. and S. F. Altschul (1990)Proc. Natl. Acad. Sci. USA 87: 2264-68; Karlin, S. and S. F. Altschul(1993) Proc. Natl. Acad. Sci. USA 90: 5873-7).

Proteins of the present invention, or proteins functionally equivalentthereto may be proteins subjected to various modifications such asphysiological modification with sugar chains and the like, labeling withfluorescence or radioactive substances, or fusion with other proteins.Particularly in recombinants that will be described later, sugar chainmodification may vary depending on the hosts used for expression.However, even if there is a difference in sugar chain modifications, allproteins having properties similar to those of HMGB1, HMGB2, HMGB3,S100A8, or S100A9 proteins disclosed herein are HMGB1, HMGB2, HMGB3,S100A8, or S100A9 proteins of the present invention or proteinsfunctionally equivalent thereto.

HMGB1, HMGB2, FIMGB3, S100A8, or S100A9 proteins can be obtained notonly from living materials, but also in the form of recombinants byincorporating genes that encode these proteins into an appropriateexpression system. In order to obtain HMGB1, HMGB2, HMGB3, S100A8, orS100A9 proteins by genetic engineering techniques, the above-mentionedDNAs which encode HMGB1, HMGB2, HMGB3, S100A8, or S100A9 proteins may beincorporated into an appropriate expression system, and they can then beexpressed. Examples of host/vector systems applicable to the presentinvention include the expression vector pGEX and E. coli. With pGEX,foreign genes can be expressed as a fusion protein withglutathione-S-transferase (GST) (Gene, 67: 31-40, 1988). pGEXincorporated with a gene encoding the HMGB1, HMGB2, HMGB3, S100A8, orS100A9 protein is introduced into an E. coli strain such as BL21 by heatshock, incubated for an appropriate time and thenisopropylthio-β-D-galactoside (IPTG) is added to induce the expressionof GST-fused HMGB1, GST-fused HMGB2, GST-fused HMGB3, GST-fused S100A8,or GST-fused S100A9 proteins. Since GST of the present invention adsorbsonto Glutathione Sepharose 4B, the expression product is readilyseparated and purified by affinity column chromatography.

In addition, the following may also be applied as host/vector systems toobtain recombinants of HMGB1, HMGB2, HMGB3, S100A8, or S100A9 proteins.First, when bacteria are used as hosts, expression vectors for fusionproteins that utilize histidine-tag, HA-tag, a FLAG-tag, and the likeare commercially available. Regarding yeasts, yeasts belonging to thegenus Pichia are known to be effective for the expression of sugarchain-containing proteins. In terms of the addition of sugar chains,expression systems that utilize baculovirus vector with insect cells asa host are also useful (Bio/Technology, 6: 47-55, 1988). Further, usingmammalian cells, transfection of a vector is carried out using promoterssuch as CMV, RSV, and SV40. Any of these host/vector systems can be usedas an expression system of HMGB1, HMGB2, HMGB3, S100A8, or S100A9proteins. Moreover, genes can also be introduced using viral vectorssuch as retrovirus vectors, adenovirus vectors, and adeno-associatedvirus vectors.

Thus obtained proteins of the present invention may be isolatedintracellularly or extracellularly (medium and such), and can bepurified as proteins that are substantially pure and homogenous.Proteins may be separated and purified using separation and purificationmethods which are commonly used in protein purification, and are notparticularly limited. For example, proteins can be separated andpurified by appropriately selecting and combining a chromatographycolumns, filters, ultrafiltration, salting out, solvent precipitation,solvent extraction, distillation, immunoprecipitation,SDS-polyacrylamide gel electrophoresis, isoelectric focusingelectrophoresis, dialysis, recrystallization, and the like.

Examples of chromatographies include affinity chromatography,ion-exchange chromatography, hydrophobic chromatography, gel filtration,reverse phase chromatography, and adsorption chromatography (Marshak etal., Strategies for Protein Purification and Characterization: ALaboratory Course Manual. Ed Daniel R. Cold Spring Harbor LaboratoryPress, 1996). These chromatographies can be performed using liquid phasechromatographies such as HPLC and FPLC.

Moreover, proteins of the present invention are preferably substantiallypurified proteins. Here, the term “substantially purified” means thatthe protein purity of the present invention (proportion of the proteinof the present invention in total protein components) is 50% or more,60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 100% orclose to 100%. The upper limit for “close to 100%” depends on thepurification techniques and analytical techniques of those skilled inthe art, of which examples are 99.999%, 99.99%, 99.9%, 99%, and thelike.

Moreover, a substantially purified protein includes any protein purifiedby any purification method as long as the protein purity is as mentionedabove. Examples include, but are not limited to, proteins substantiallypurified by appropriately selecting and combining the above-mentionedchromatography columns, filters, ultrafiltration, salting out, solventprecipitation, solvent extraction, distillation, immunoprecipitation,SDS-polyacrylamide gel electrophoresis, isoelectric focusingelectrophoresis, dialysis, recrystallization, and the like.

Cells where HMGB1, HMGB2, HMGB3, S100A8, or S100A9 proteins of thepresent invention are released or secreted basically include all typesof tissue-derived cells in vivo. Cells which can be readily collectedand cultured are exemplified by, but are not limited to, fibroblasts(such as normal skin fibroblasts and cell lines derived therefrom).Moreover, cells secreting HMGB1, HMGB2, HMGB3, S100A8, or S100A9proteins can also be produced by the following manner. A vector isproduced by inserting an HMGB1, HMGB2, HMGB3, S100A8, or S100A9protein-encoding DNA, or an HMGB1, HMGB2, HMGB3, S100A8, or S100A9protein-encoding DNA linked with a secretion signal-encoding DNA (ATGCAG ACA GAC ACA CTC CTG CTA TGG GTA CTG CTG CTG TGG GTT CCA GGT TCC ACTGGT GAC; SEQ ID NO: 29), into a known expression vector or a genetherapy vector. The produced vector is introduced into mammalian cellssuch as fibroblasts (such as normal skin fibroblasts and cell linesderived therefrom), insect cells, and other cells. Examples of secretionsignal-encoding DNAs include, but are not limited to, DNAs with theabove-described sequences. Furthermore, there are no particularlimitations in the animal type from which these cells derive, althoughcells from the animal type of the target animal subjected to vectoradministration, cells from the target itself, or cells derived from ablood relative of the target subjected to vector administration arepreferably used.

DNAs which encode HMGB1, HMGB2, HMGB3, S100A8, or S100A9 proteins of theinducers or tissue regeneration promoters of the present invention maybe cDNAs, genomic DNAs, natural DNAs, or artificially-synthesized DNAsso long as they encode the HMGB1, HMGB2, HMGB3, S100A8, or S100A9protein. DNAs which encode HMGB1, HMGB2, HMGB3, S100A8, or S100A9proteins are normally administered in a form inserted in vectors.

Examples of the vectors of the present invention include, but are notlimited to, plasmid vectors, retrovirus vectors, lentivirus vectors,adenovirus vectors, adeno-associated virus vectors, Sendai virusvectors, Sendai virus envelope vectors, and papilloma virus vectors. Thevectors may contain promoter DNA sequences which effectively induce geneexpression, factors that regulate gene expression, and molecules whichare necessary for maintaining DNA stability.

In the present invention, the following vectors may also be used:partial peptides of HMGB1, HMGB2, HMGB3, S100A8, or S100A9 protein whichhave an activity of inducing bone marrow-derived cells; cells secretingthese partial peptides; or vectors inserted with the DNAs encoding thesepartial peptides.

Hyaluronic acid (also called hyaluronan) is a glycosaminoglycan(mucopolysaccharide) in which the two sugars, N-acetylglucosamine andglucuronic acid, are connected and linked. The chemical name is:[→3)-2acetamido-2deoxy-β-D-glucopyranosyl-(1→4) β-D-glucopyranosyluronicacid-(1→]n. Most natural hyaluronic acids are high-molecular-weighthyaluronic acids with a molecular weight of several hundred thousands.Meanwhile, low-molecular-weight hyaluronic acids with two to 14 sugarscan be artificially produced. In the medical field, for example, sodiumhyaluronate has been used for injection into the joint cavity. Methodsfor producing hyaluronic acids include methods in which hyaluronic acidsare extracted and purified from products produced using Streptococcuszooepidemicus, a lactic acid bacterium, and methods in which hyaluronicacids are extracted and purified from chicken crest or such. Hyaluronicacids can be variously modified by esterification, periodate oxidation,isourea coupling, sulfation, and such. Hyaluronic acids can alsocrosslinked via ether or ester bridges. Such modifications stabilizehyaluronic acids against degradation, insolubilize them, make them intoa spongy form, or confer them with the property to release substances ina sustained fashion. Meanwhile, CD44 is a hyaluronic acid receptor, andhyaluronic acids have chemotactic activity for mesenchymal stem cellsthat have CD44 on their cell surface.

Extracts of cells or tissues used in the methods of the presentinvention can be produced by methods comprising the step of immersingcells or tissues in a solvent.

Cells and tissues to be immersed in a solvent are not particularlylimited, but include, for example, tissue-derived cells, cells of celllines established from tissue-derived cells (including, but not limitedto, for example, HeLa and HEK293), isolated cells, non-isolated cells(for example, cells in isolated tissues), and cells transfected with DNAencoding HMGB1, HMGB2, HMGB3, S100A8, or S100A9 protein. The abovetissues may be any types of tissue, and include, but are not limited to,for example, live skin tissues and tissues obtained by biopsy (surgery)from the body (brain, lung, heart, liver, stomach, small and largeintestines, pancreas, kidney, urinary bladder, spleen, uterus, testis,blood, etc.).

Examples of the above solvent include, but are not limited to,physiological saline, phosphate-buffered saline (PBS), and Tris-bufferedsaline (TBS). Moreover, the immersion time of cells or tissue in asolvent should be a duration necessary and sufficient for inducing cellnecrosis, that is, 1 hour to 48 hours (such as 6 to 48 hours), andpreferably 12 to 24 hours, but is not limited thereto. Therefore, the“step of immersing cells in a solvent” can be rephrased as a “step ofimmersing cells in a solvent for a duration necessary and sufficient forinducing necrosis” or “step of necrosing cells”. Moreover, examples ofthe temperature for immersing cells or tissue in a solvent include, butare not limited to, 4° C. to 25° C. (such as 4° C. to 8° C.), andpreferably 4° C. Further, examples of the pH for immersing cells ortissue in a solvent include, without limitation, pH 7 to 8, andpreferably pH 7.5. Examples of the buffer include, without limitation, aphosphate buffer solution at a concentration of 10 mM to 50 mM,preferably 10 to 20 mM, but are not limited thereto.

Moreover, in the present invention, cells or tissues can be removed froma solvent containing them after they are immersed in the solvent. Themethod for removing cells or tissues from a solvent is not particularlylimited as long as the method is well known to those skilled in the art.For example, cells or tissues can be removed from a solvent bycentrifuging at a gravity acceleration of 10 G to 100,000 G (forexample, 440 G) at 4° C. to 25° C. (for example, 4° C.), followed byseparation of the supernatant, but the removal method is not limitedthereto. The supernatant can be used as an extract of cells or tissues.

The extracts of cells or tissues of the present invention prepared bymethods comprising the step of immersing cells or tissues in a solventinclude, for example, skin extract and peripheral blood mononuclear cellextract (peripheral blood extract), but are not limited thereto.

The peripheral blood extract is prepared by the following method: aftercollecting blood with a syringe or the like, the cells are frozen in afreezer or liquid nitrogen, on dry ice, or such, and then thawed at atemperature of 0° C. or higher. Then, to remove insoluble cellularcomponents, the sample is centrifuged, for example, at a gravity of 10to 100,000 G (for example, at 440 G) and 4° C. to 25° C. (for example,at 4° C.), and the resulting supernatant is collected. The insolublecellular components can be removed from the solvent by the methoddescribed above. However, methods for removing insoluble cellularcomponents are not limited to the above example. The resultingsupernatant can be used as an extract of cells or tissues.Alternatively, instead of centrifugation, insoluble cellular componentscan be removed by filtration through a nitrocellulose filter with micropores of 0.45 μm, or the like. Alternatively, collected peripheral bloodmay be allowed to stand for three to 48 hours at 4° C. to induce cellnecrosis. The intracellular components can be released from peripheralblood cells by this treatment. Then, to remove insoluble cellularcomponents from the solvent, the sample is centrifuged at a gravity of10 to 100,000 G (for example, at 440 G), and the resulting supernatantis collected. The insoluble cellular components can be removed from thesolvent by the method described above, but are not limited thereto. Theresulting supernatant can be used as an extract of cells or tissues.Alternatively, instead of centrifugation, insoluble cellular componentscan be removed by filtration through a nitrocellulose filter with micropores of 0.45 μm of the like.

Heparin-binding fractions from the extracts of cells or tissues to beused in the present invention can be produced by a method comprising thefollowing steps.

(a) immersing a cell or tissue in a solvent;

(b) contacting an extract obtained by the step (a) with immobilizedheparin; and

(c) eluting a heparin-binding fraction (may also be expressed asheparin-purified fraction or heparin-column purified fraction) from theimmobilized heparin.

“Immobilized heparin” refers to heparin covalently bound to an insolublecarrier. Examples of the insoluble carrier include, but are not limitedto, Sepharose beads (such as Sepharose 4B, Sepharose 6B and such: GEHealthcare). In the present invention, a commercially availableimmobilized heparin (Hitrap Heparin HP column: GE Healthcare) may alsobe used.

Examples of conditions for contacting an extract of cells or tissueswith immobilized heparin include, but are not limited to, about pH 7 to8 (preferably pH 7.5), and a salt concentration of 0 to 200 mM, andpreferably about 100 to 200 mM. The time the extract is in contact withimmobilized heparin is not specifically limited, but the contact ispreferably retained for 5 minutes or more in view of sufficientadsorption of the heparin-binding fraction onto immobilized heparin.Examples of the temperature include, but are not limited to, 4 to 8° C.,and preferably 4° C. Further, examples of the elution condition of theheparin-binding fraction adsorbed onto the immobilized heparin include,but are not limited to, a pH of about 7 to 8 and a salt concentration of200 to 1,000 mM (preferably about 1,000 mM).

Meanwhile, any of the materials of (a) to (r) described above ormixtures of any two or more of the materials, which are used in themethods of the present invention, include but are not limited to, forexample, the combinations of:

-   -   hyaluronic acid and HMGB1 protein; hyaluronic acid and HMGB2        protein; hyaluronic acid and HMGB3 protein; hyaluronic acid and        S100A8 protein; hyaluronic acid and S100A9 protein;        hyaluronic acid, and HMGB1 and HMGB2 proteins; hyaluronic acid,        and HMGB2 and HMGB3 proteins; hyaluronic acid, and HMGB1 and        HMGB3 proteins; hyaluronic acid, and HMGB1, HMGB2, and HMGB3        proteins; hyaluronic acid, and S100A8 and S100A9 proteins;        hyaluronic acid, and HMGB1 and S100A8 proteins; hyaluronic acid,        and HMGB2 and S100A8 proteins; hyaluronic acid, and HMGB3 and        S100A8 proteins; hyaluronic acid, and HMGB1 and S100A9 proteins;        hyaluronic acid, and HMGB2 and S100A9 proteins; hyaluronic acid,        and HMGB3 and S100A9 proteins; hyaluronic acid and extracts of        cells or tissues; and hyaluronic acid and heparin-binding        fractions of cell or tissue extracts; and preferably mixtures of        hyaluronic acid, and extracts of cells or tissues. The mixing        ratio is based on the volume of the least amount of ingredient        dissolved in a solvent which is taken as 1, where the other        ingredients may be added in a volume up to 10,000 times greater,        and preferably, the other ingredients may be added in a volume        equal or up to 10 times greater when the least amount of        ingredient is taken as 1. The types of animals which serve as a        source for an extract, heparin-binding fraction, HMGB1, HMGB2,        HMGB3, S100A8, or S100A9 protein of the present invention        include human and non-human animals, which can be exemplified by        humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters, and        guinea pigs, but the type of animal is preferably the same as        the animal to be administered with the above extract or the        like.

Extracts, heparin-binding fractions, HMGB1, HMGB2, HMGB3, S100A8, andS100A9 proteins of the present invention are administered to bloodvessels or muscles by parenteral administration methods. Specifically,such administration methods include injection. For example, thepharmaceutical agents of the present invention can be administered toblood vessels, muscles, or under the skin (for example, into or near thevessel implanted under the skin) by intravascular injection(intraarterial injection, intravenous injection, etc.), intramuscularinjection, subcutaneous injection, and the like. Alternatively, theagents of the present invention may be allowed to absorb in asustained-release manner using transdermal absorbable patches orexternal preparations.

The method of administration may be appropriately selected according tothe age and the symptoms of the subject. When an HMGB1, HMGB2, HMGB3,S100A8, or S100A9 protein is administered, the dose per time of theprotein can be selected within a range of 0.0000001 mg to 1,000 mg perkg body weight of a subject. Alternatively, the dose can be selectedwithin a range of 0.00001 mg to 100,000 mg per body of subject, forexample. When administering cells secreting HMGB1, HMGB2, HMGB3, S100A8,or S100A9 proteins or gene therapy vectors inserted with DNAs encodingHMGB1, HMGB2, HMGB3, S100A8, or S100A9 proteins they may be administeredsuch that the amounts of HMGB1, HMGB2, HMGB3, S100A8, or S100A9 proteinin the damaged tissues are within the above range. However, the dosageof extracts, heparin-binding fractions, or HMGB1, HMGB2, HMGB3, S100A8,or S100A9 proteins of the present invention is not limited thereto.

When administered, extracts, heparin-binding fractions, or HMGB1, HMGB2,HMGB3, S100A8, or S100A9 proteins can be formulated according to theusual methods (for example, Remington's Pharmaceutical Science, latestedition, Mark Publishing Company, Easton, U.S.A), and may containpharmaceutically acceptable carriers and additives together. Examplesinclude surfactants, excipients, colorants, perfumes, preservatives,stabilizers, buffers, suspending agents, isotonizing agents, binders,disintegrants, lubricants, flow promoters, and flavoring agents,although they are not limited thereto and other common carriers may beappropriately used. Specific examples include light anhydrous silicicacid, lactose, crystalline cellulose, mannitol, starch, carmellosecalcium, carmellose sodium, hydroxypropylcellulose,hydroxypropylmethylcellulose, polyvinylacetaldiethylamino acetate,polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride,polyoxyethylene hydrogenated castor oil 60, white sugar, carboxymethylcellulose, corn starch, and inorganic salts.

Extracts, heparin-binding fractions, HMGB1, HMGB2, HMGB3, S100A8, orS100A9 protein of the present invention can be placed into a vessel byinjection using the following method. Specifically, each solution orsolute is dissolved in physiological saline, and the resulting solutionis manually injected using an injector or pipette. Alternatively, thesolution is mechanically injected into a tube at the time of tubeproduction.

Meanwhile, the amount of extracts, heparin-binding fractions, HMGB1,HMGB2, HMGB3, S100A8, or S100A9 protein of the present invention to beinjected into a vessel is determined appropriately according to thevolume of the vessel used for cell collection. For example, when thevessel is implanted into abdominal subcutaneous adipose, it is possibleto inject about 10 ml of an agent of the present invention.Alternatively, when the vessel is implanted under non-abdominal skin,the amount of injection is expected to be approximately severalmilliliters.

The present invention provides cell populations collected by theabove-described methods for harvesting cell population.

The present invention also provides bone marrow cells collected by theabove-described methods for harvesting bone marrow cells.

Cell populations and bone marrow cells of the present invention can beadministered to treat diseases with tissue damage such as hereditarydiseases, skin diseases (thermal injury, cutaneous ulcer, or such),brain and nerve diseases (brain infarction, Alzheimer's disease, spinalcord injury, brain injury, or such), cardiovascular diseases (myocardialinfarction, cardiac myopathy, arterial embolism, or such), andosteocartilaginous diseases (bone fracture, rheumatism, or such).Tissues can be regenerated by directly administering the cells intoblood circulation via a vein or artery, or by directly administering thecells into tissues at the damaged site immediately after cell harvest.Alternatively, tissues can be regenerated by administering cells in adispersed or aggregated state, or when they are formed in a sheet shapeafter the cells are cultured using culture dishes or flasks. With regardto the amount of administration, 1 to 10¹⁴ cells, and preferably 10² to10¹⁰ cells can be administered. Thus, the present invention alsoprovides tissue-regenerating agents comprising a cell populationharvested by the above-described methods for harvesting cell populationsor bone marrow cells harvested by the above-described methods forharvesting bone marrow cells.

The type of tissues to be regenerated is not particularly limited. Thetarget tissue may be any tissue, as long as it is a damaged tissue. Suchtissues include, for example, live skin tissues and tissues obtained bybiopsy (surgery) from the body (brain, lung, heart, liver, stomach,small and large intestines, pancreas, kidney, urinary bladder, spleen,uterus, testis, blood, and such). In particular, the agents of thepresent invention are effectively used to regenerate tissues that aredifficult for direct administration of agents from outside of the body(brain, heart, or such). In the present invention, damaged tissuesinclude tissues damaged due to various pathological conditions such asthose that result in ischemia, hemostasis or hypoxia, trauma, burn,inflammation, autoimmunity, genetic abnormalities, and such, but are notlimited to these examples. Furthermore, damaged tissues include necrotictissues.

Tissues of the present invention are not particularly limited, as longas they are differentiable from bone marrow cells. Such tissues include,for example, all biological tissues such as skin tissues, bone tissues,cartilage tissues, muscle tissues, adipose tissues, myocardial tissues,nerve tissues, lung tissues, gastrointestinal tissues, liver tissues,gallbladder tissues, pancreatic tissues, and urogenital system.Furthermore, the above-described tissue-regenerating agents can be usedto treat not only skin diseases such as intractable cutaneous ulcer,skin wounds, bullosis, and alopecia, but also tissue damages such asthose that are due to brain infarction, myocardial infarction, bonefracture, pulmonary infarction, gastric ulcer, and enteritis. Thespecies of animals to be administered with the above-describedtissue-regenerating agents include, but are not limited to human andnon-human animals, for example, humans, mice, rats, monkeys, pigs, dogs,rabbits, hamsters, and guinea pigs. Furthermore, the agents of thepresent invention can be administered to diabetes patients. It is knownthat intractable cutaneous ulcer which is a complication of diabetes isdifficult to treat as compared to cutaneous ulcer in normal persons. Theagents of the present invention are also effectively used for suchdiabetes patients.

Following cell harvest, the cells are diffused in physiological saline,and the resulting cell suspension can be administered as atissue-regenerating agent of the present invention into circulatoryblood flow such as a vein or artery to achieve tissue regeneration.Alternatively, the tissue-regenerating agent of the present inventionmay be applied or plastered onto the surface of a tissue damage site toachieve tissue regeneration. Alternatively, the agent may be injectedinto the damaged site using a syringe or the like to achieve tissueregeneration. Alternatively, the cells may be cultured using culturedishes or flasks, and then administered to the damaged site in adiffused or aggregated state, or when they are formed in a sheet shape,to achieve tissue regeneration. With regard to the amount ofadministration, 1 to 10¹⁴ cells, and preferably 10² to 10¹⁰ cells can beadministered.

All prior art documents cited herein are incorporated by referenceherein.

EXAMPLES

Hereinbelow, the present invention will be specifically described withreference to the Examples, but it is not to be construed as beinglimited thereto.

Example 1

Purpose: To develop novel highly efficient and minimally invasivetechniques for collecting functional cells such as stem cells whichexist in an extremely low number in vivo.

Methods: Studies were conducted to achieve the above purpose.

(1) After irradiating at 10 Gy, eight-week-old mice (male) weretransplanted via the caudal vein with bone marrow cells (5×10⁶ cells/0.1ml of phosphate buffered saline, pH 7.4) derived from green fluorescentprotein (GFP) transgenic mice to prepare GFP bone marrow-transplantedmice.(2) Tubes made of biologically hypoallergenic material (silicone) werefilled with HMGB1, hyaluronic acid, skin extract, or phosphate bufferedsaline (pH 7.4), and then implanted under the dorsal skin of GFP bonemarrow-transplanted mice (FIG. 1).(3) The implanted tubes were removed two weeks after implantation (FIG.2). Cells accumulated in the tubes (tube-entrapping cells (TECs)) werecollected and then cultured in Dulbecco's medium (D-MEM) supplementedwith 10% fetal bovine serum at 37° C. under 5% carbon dioxide gas.(4) When the culture density of TECs reached about 80% confluent on thebottom surface of culture dish, the culture medium was changed with abone differentiation-inducing medium, adipose differentiation-inducingmedium, or epidermis differentiation-inducing medium. Then, the cellswere further cultured. Two weeks after the start of culture in eachdifferentiation-inducing medium, differentiation into bone, adipose, andepidermis was assessed by alizarin red staining, oil red staining, andimmunostaining for keratin 5, respectively.(5) The proportion of platelet-derived growth factor receptor a and CD44double-positive cells to TECs was analyzed by flow cytometry.

Results: Silicone tubes filled with HMGB1, hyaluronic acid, or skinextract were implanted under the skin of GFP bone marrow-transplantedmice. As a result, when the tubes were filled with any of thesesolutions, a large number of GFP-positive bone marrow-derived cells(TECs) were accumulated in the tubes in one week. An image ofGFP-positive cells accumulated in a silicone tube filled with HMGB1 isshown in FIG. 2. Meanwhile, the number of adhesive TECs in the siliconetubes filled with phosphate buffer was significantly smaller. When cellscollected from silicone tubes filled with HMGB1 were cultured,proliferating adherent TECs were observed to be adhered to the culturedish 24 hours after the start of culture (FIGS. 3A-3B). Adherent TECsthat had migrated into silicone tubes filled with HMGB1 were cultured ina bone differentiation-inducing medium, adipose differentiation-inducingmedium, or epidermis differentiation-inducing medium. The result showedthat the cells had the ability to differentiate into alizarinred-positive osteoblasts, oil red-positive adipocytes, and keratin5-positive epidermal cells, and thus among adherent TECs exist cellpopulations capable of mesenchymal and epithelial differentiation (FIGS.4A-6). Furthermore, the expression of PDGFRα and CD44 on TECs wasassessed by flow cytometry. PDGFRα and CD44 are known to be expressed onthe surface of mesenchymal stem cells. The result showed that about 60%of TECs were positive for both PDGFRα and CD44 (FIG. 7).

In addition, tubes filled with hyaluronic acid at 100 ng/ml (in PBS)were implanted under the dorsal skin of GFP bone marrow-transplantedmice. After two weeks, the tubes were removed and cells mobilized intothe tubes were analyzed for their surface markers by FACS. The resultswere similar to that obtained with TECs isolated from HMGB1 tubes, anddemonstrated that about 60% of the cells were P44 cells positive forboth CD44 and PDGFRα and had the ability to differentiate intomesenchymal cells and epithelial cells such as osteoblasts andadipocytes.

Discussion: In the present invention, the present inventors revealedthat bone marrow-derived TECs having the ability to differentiate intobone, adipose, and epidermis could be very efficiently collected byimplanting under the skin, silicone tubes filled with HMGB1, hyaluronicacid, or skin extract. The following findings suggest that mesenchymalstem cells are selectively mobilized to TECs: most of the bonemarrow-derived TECs express PDGFRα and CD44 which are known to beexpressed on the surface of mesenchymal stem cells; and HMGB1,hyaluronic acid, and skin extract all have the activity of mobilizingmesenchymal stem cells. By selecting a solution for filling the siliconetube, specific biologically functional cells can be selectivelyharvested with high efficiency according to the substance contained inthe solution, which is responsible for mobilizing the specificbiologically functional cells. This novel technique of the presentinvention enables one to collect desired biologically functional cellswithout using highly invasive methods such as inserting a needle intothe bone marrow. Thus, the technique is expected to enable the designingof order-made regenerative medical techniques according to the purpose.Furthermore, collected cells can be provided as materials for varioustypes of basic studies including stem cell research. Thus, the presentinventors believe that the present invention contributes largely to thedevelopment of basic and clinical studies, as well as drug discovery andprogress of medical technologies.

Example 2

Purpose: To assess the therapeutic effect of bone marrow-derived cellsmobilized into hypodermal device on skin ulcer

Methods:

(1) Devices to be implanted into the body were prepared.

(2) To prepare skin tissue extract containing factors that mobilize bonemarrow pluripotent stem cells, free skin pieces were isolated from twoheads of C57/BL6 neonatal mice (two-day-old) and immersed in 2 ml ofphosphate buffered saline (PBS), pH 7.4. After 24 hours of incubation at4° C., the sample was centrifuged at 440 G and 4° C. for ten minutes toremove the tissues. The resulting supernatant was collected as skinextract. Furthermore, to prepare peripheral blood mononuclear cellextract, peripheral whole blood was collected from two heads of4-week-old C57/BL6 mice and then diluted with PBS to a total volume of 4ml. After placing 3 ml of Ficoll-Paque Plus (GE) in a centrifuge tube,the diluted blood was overlaid onto the Ficoll layer. The tube wascentrifuged at 100 G at 18° C. for ten minutes. The resulting middlelayer containing mononuclear cells was transferred into a freshcentrifuge tube. To remove Ficoll-Paque Plus from the collected middlelayer, 45 ml of PBS was added and the resulting mixture was centrifugedat 440 G at 18° C. for five minutes. The supernatant was removed, andagain 45 ml of PBS was added, and the suspension was centrifuged at 440G at 18° C. for five minutes. The supernatant was removed, and theprecipitated cells were suspended in 200 μl of PBS. The cell suspensionwas frozen in a freezer at −80° C. for 30 minutes, and then transferredonto ice for thawing. This freeze-thaw treatment was repeated threetimes. The sample was centrifuged at 440 G at 4° C. for 15 minutes. Theresulting supernatant was collected (peripheral blood extract).

(3) Male C57/BL6 mice (six to eight weeks old) were irradiated at alethal dose (10 Gy). Immediately after irradiation, green fluorescentprotein (GFP) transgenic mouse-derived bone marrow cells (5×10⁶cells/0.1 ml of phosphate buffered saline, pH 7.4) were transplanted tothe mice via the caudal vein. Only mice that had survived for eightweeks after transplantation were used in subsequent experiments.

(4) 40 μl of the skin extract and peripheral blood extract prepared asdescribed in (2), or PBS as a negative control were each inserted intothe devices. A pair of devices was implanted into the mice as preparedin (3) (a total of two devices per head). The devices were placed underthe dorsal skin: one was placed on the right and the other on the left,arranged so that their openings were in contact with the fascia. Twoweeks after implantation, the devices were removed from the mice.

(5) After removing dorsal hair from eight-week-oldC.B-17/ler-scid/scidJcl (Charles River Japan, Inc), round-shapedcutaneous ulcers with a diameter of 6 mm were formed on either side ofthe back. To prevent shrinkage of mouse skin, a silicone disc with anouter diameter of 10 mm, inner diameter of 6 mm, and thickness of 1 mmwas adhered to the ulcer site using two-sided adhesive tape and medicaladhesive, Aron alpha A (Sankyo). Cells were collected from the inside ofeither one of the left and right devices removed as described in (3) andadministered to the cutaneous ulcer. A silicone disc with a diameter of10 mm and thickness of 1 mm was placed over the ulcer to preventdesiccation and bacterial infection. Then, the ulcer was covered withTegaderm (3M) for protection. The area of the ulcer was measured afterseven days (FIG. 27).

As a result, the negative control ulcer was not closed at the surfaceeven after seven days. Meanwhile, in the ulcer administered with cellscollected with the blood extract, reduction of the ulcer surface areawas observed on day 5. On the other hand, when administered with cellscollected using the skin extract, the ulcer was found to be closed onthe surface on day 7 (FIG. 27).

This example demonstrated that when administered to cutaneous ulcerlesions, bone marrow-derived cells mobilized into an in vivoimplantation device that contains tissue extract produced a therapeuticeffect on the ulcer. The therapeutic method of the present inventionwhich uses bone marrow cells mobilized into implanted devices but doesnot collect bone marrow cells from the bone marrow is an entirely noveltherapeutic method.

Example 3

(1) Skin extract and peripheral blood extract were prepared by themethod described above in Example 2.

1-ml HiTrap Heparin HP columns (GE) were equilibrated with 10 ml of 10mM phosphate buffer (pH 7.5). Each skin extract and peripheral bloodextract was diluted with ten volumes of 10 mM phosphate buffer (pH 7.5)and loaded onto equilibrated columns. To wash off non-specificallyadsorbed materials, the columns were washed with 10 ml of 10 mMphosphate buffer (pH 7.5). The adsorbed components were eluted with 10mM phosphate buffer (pH 7.5) containing 1,000 mM NaCl, and aliquoted(120 μl) into plastic tubes. The protein contents were determined usinga protein assay kit (Bio-Rad), and three fractions with the highestprotein concentration were recovered (heparin-adsorbed tissue extract).

(2) 10 μl of PBS containing 0.1% hyaluronic acid was combined with 40 μlof the tissue extract. The resulting mixture was added into the devicesdescribed in Example 2. A mixture of hyaluronic acid and 10 mM phosphatebuffer (pH 7.5) containing 1,000 mM NaCl was used as negative control.

(3) GFP bone marrow-transplanted mice were prepared by the same methodas described in Example 2. The devices prepared as described in (2) wereimplanted under the dorsal skin of the mice. Ten days afterimplantation, the devices were removed from under the skin and cellswere collected from the devices. The collected cells were cultured inculture dishes containing IMDM (Invitrogen) supplemented with 10% fetalbovine serum at 37° C. under an atmosphere of 5% CO₂. To removenon-adherent cells, the medium was changed one day after the start ofculture. Then, the medium was changed every three days. One week aftercell harvest, the cells were observed under a fluorescence microscope todetect bone marrow-derived cells (GFP-positive cells) (FIGS. 28A-28D).Furthermore, the number of GFP-positive cells was determined using animage analysis software (Image J) (FIG. 29).

The mixture of hyaluronic acid and heparin-binding fraction of tissueextract (blood extract (FIG. 28C), and skin extract (FIG. 28D)) weredemonstrated to have a strong activity of mobilizing bone marrow-derivedadherent cells into the device (FIGS. 28A-28D and 29), as compared toPBS (FIG. 28A) or hyaluronic acid alone (FIG. 28B).

This Example demonstrates that bone marrow cell-mobilizing factors canbe purified from tissue extracts using heparin columns. Skin extractcontains HMGB1, HMGB2, HMGB3, S100A8, and S100A9. These components bindto heparin columns, suggesting the possibility that these cells areinvolved in the mobilization of bone marrow cells. Alternatively, sincevarious cytokines such as growth factors also bind to heparin columns,it is thought that the net effect of the various factors is responsiblefor the mobilization of bone marrow cells.

Example 4

(1) RNA was extracted from neonatal mouse skin using Trizol(Invitrogen), and then cDNA was synthesized from the RNA using theSuperScript III cDNA Synthesis Kit (Invitrogen). S100A8 cDNA wasamplified by the polymerase chain reaction (PCR) method using thesynthesized cDNA as a template. For purification, S100A8 cDNA wasinserted into the mammalian cell protein-expression plasmid vectorpCAGGS to be expressed as a protein having Flag-tag and 6×His-tagsequences at the N terminus of the amino acid sequence.pCAGGS-Flag-His-S100A8 was transfected into a human fetal kidneycell-derived cultured cell line HEK293 using polyethyleneimine (PEI).After 48 hours, the cells and culture supernatant were separatelycollected by centrifugation at 4,400 G and 4° C. for five minutes. Then,the collected supernatant was filtered through a cellulose acetatefilter having pores with a diameter of 0.8 μm (Nalgene) and then througha nitrocellulose filter having pores with a diameter of 0.45 μm(Corning) to prepare a sample removed of insoluble fractions. The samplewas loaded onto 5-ml HisTrap FF (GE) equilibrated with 50 ml of 50 mMTris HCl (pH 8.0) containing 50 mM NaCl, and then the adsorbedcomponents were washed with 50 mM Tris HCl (pH 8.0) containing 50 mMNaCl and 10 mM imidazole to remove nonspecifically adsorbed components.The adsorbed specific components were eluted from the column using 50 mMTris HCl (pH 8.0) containing 50 mM NaCl and 100 mM imidazole. Theadsorbed fractions were fractionated into silicone-coated plastic tubes(500 μl/tube). Protein-containing fractions were combined together andmixed with anti-Flag Antibody M2 Beads (Sigma). The mixtures wereincubated at 4° C. for 12 hours with gentle mixing. After incubation,the beads were centrifuged at 440 G for five minutes. After removing thesupernatant, the beads were resuspended in PBS and centrifuged in thesame way. The supernatant was removed. The beads were loaded onto a 3-mlcolumn with an inner diameter of 1 cm, and the adsorbed protein waseluted from the column using 100 mM glycine (pH 3.5). The eluate wasneutralized with a 1/10 volume of 500 mM Tris HCl (pH 7.5). The purifiedprotein was quantified using a protein assay kit (Bio-Rad).

(2) RNA was extracted from neonatal mouse skin using Trizol(Invitrogen), and then cDNA was synthesized from the RNA using theSuperScript III cDNA Synthesis Kit (Invitrogen). HMGB1 cDNA wasamplified by the polymerase chain reaction (PCR) method using thesynthesized cDNA as a template. For purification, HMGB1 cDNA wasinserted into the mammalian cell protein-expression plasmid vectorpCAGGS to be expressed as a protein having Flag-tag and 6×His-tagsequences at the N-terminus of the amino acid sequence.pCAGGS-Flag-His-S100A8 was transfected into a human fetal kidneycell-derived cultured cell line HEK 293 using polyethyleneimine (PEI).After 48 hours, the cells and culture supernatant were separatelycollected by centrifuging at 4,400 G at 4° C. for five minutes. Then,the collected supernatant was filtered through a cellulose acetatefilter having pores with a diameter of 0.8 μm (Nalgene) and then througha nitrocellulose filter having pores with a diameter of 0.45 μm(Corning) to prepare a sample removed of insoluble fractions. The samplewas loaded onto 5-ml HisTrap FF (GE) equilibrated with 50 ml of 50 mMTris HCl (pH 8.0) containing 50 mM NaCl, and then the absorbedcomponents were washed with 50 mM Tris HCl (pH 8.0) containing 50 mMNaCl and 10 mM imidazole to remove nonspecifically adsorbed components.The adsorbed specific components were eluted from the column using 50 mMTris HCl (pH 8.0) containing 50 mM NaCl and 100 mM imidazole. Theadsorbed fractions were fractionated into silicone-coated plastic tubes(500 μl/tube). Protein-containing fractions were combined together, andthen imidazole was removed using a desalting column PD10 (GE). Thefractions were eluted using 50 mM Tris HCl (pH. 7.5) containing 150 mMNaCl. HRV3C (Novagen) was added to the eluted samples and the mixturewas incubated at 4° C. for three hours. After cleavage, the sample wasloaded onto a 1-ml HiTrap Heparin column (GE) equilibrated with 50 mMTris HCl (pH 7.5) containing 150 mM NaCl. The inside of the column waswashed with 50 mM Tris HCl (pH 7.5) containing 150 mM NaCl. The proteinbound to the column was eluted with 50 mM Tris HCl (pH 7.5) containing1,000 mM NaCl. The eluted sample was aliquoted to silicone-coatedplastic tubes (500 μl/tube).

(3) 40 μg each of S100A8, HMGB1, HMGB2, and HMGB3 were added intosilicone devices. The devices were implanted under the right and leftdorsal skin so that the openings of devices were in contact with thefascia. Ten days after implantation, the devices were removed from underthe skin, and cells accumulated in the devices were collected from thedevices. The cells were cultured in culture dishes containing IMDM(Invitrogen) supplemented with 10% fetal bovine serum at 37° C. under anatmosphere of 5% CO₂. To remove non-adherent cells, the medium waschanged one day after the start of culture. Then, the medium was changedevery three days. One week after cell collection, the cells wereobserved under a fluorescence microscope to detect bone marrow-derivedcells (GFP-positive cells) (FIGS. 30A-30E).

(4) Round-shaped cutaneous ulcers with a diameter of 6 mm were formed onthe back (left and right sides) of eight-week-old BALB/cAJcl-nu/nu(Charles River Japan, Inc). To prevent shrinkage of the mouse skin, asilicone disc with an outer diameter of 10 mm, inner diameter of 6 mm,and thickness of 1 mm was adhered to the ulcer site using two-sidedadhesive tape and medical adhesive, Aron alpha A (Sankyo).

(5) The cells described in (3) were detached from the culture dishes bytreatment with a solution containing 0.5 g/l Trypsin and 0.53 mmol/1EDTA (Nacalai Tesque). After inactivating trypsin with IMDM containing10% FBS, the cells were centrifuged at 440 G at 4° C. for five minutes.After removing the supernatant, the cells were administered to the ulcerformed as described in (4). A silicone disc with a diameter of 10 mm andthickness of 1 mm was placed over the ulcer to prevent desiccation andbacterial infection at the site. Then, the ulcer was also covered withTegaderm (3M) for protection. The area of the ulcer was measured sevendays after administration (FIG. 31).

All of S100A8, HMGB1, HMGB2, and HMGB3 exhibited the activity ofmobilizing bone marrow cells (FIG. 30A, S100A8; FIG. 30B, HMGB1; FIG.30C, HMGB2; FIG. 30D, HMGB3) as compared to the negative control (FIG.30E). In particular, HMGB1 and HMGB2 showed stronger mobilizingactivity.

Furthermore, cutaneous ulcer was treated by administering bonemarrow-derived cells mobilized by HMGB1, HMGB2, or S100A8. The effect ofreducing ulcer (FIG. 31) was observed as compared to the negativecontrol (PBS-administered).

This Example demonstrated that all of S100A8, HMGB1, HMGB2, and HMGB3exhibited the activity of mobilizing bone marrow-derived adherent cellsinto the device. Major cells in the bone marrow are hematopoietic cellssuch as erythrocytes and hemocytes and most of the cells arenon-adherent cells. Meanwhile, mesenchymal cells such as mesenchymalstem cells are known as adherent cells. Such cells are speculated toinclude pluripotent cells capable of differentiating into epithelial andnerve cells. Since this Example demonstrated that cells mobilized intothe device using S100A8, HMGB1, or HMGB2 exerted therapeutic effect oncutaneous ulcer, the cells mobilized into the devices are bonemarrow-derived cells capable of inducing healing of tissue damages. Ithas been reported that bone marrow mesenchymal stem cells producedtherapeutic effect when administered to lesions of cutaneous ulcer(Mesenchymal stem cells enhance wound healing through differentiationand angiogenesis. Wu Y, Chen L, Scott P G, Tredget E E. Stem Cells 2007Oct.; 25(10): 2648-59; Epub 2007 Jul. 5). It is thus suggested that bonemarrow mesenchymal stem cells are involved in the therapeutic effectdescribed in this Example. Furthermore, bone marrow-derived cells suchas bone marrow mesenchymal stem cells are known to differentiate intonerve cells, adipocytes, osteocytes, chondrocytes, and epithelial cells.Cells collected by using the devices of the present invention areapplicable to novel regeneration-inducing medicine where damaged tissuesare treated by supplying the tissues with the cells.

Reference Example 1

Objective: Identification of the HMGB1 family in the skin extract andexamination of bone marrow mesenchymal stem cell-inducing activity

Methods: Whether or not the neonatal mouse skin extract contained theHMGB protein family was confirmed using the Western blot method. Freeskin pieces from 400 neonatal mice were immersed in 400 ml ofphysiological phosphate buffer solution (PBS; pH 7.4) and the solutionwas incubated at 4° C. for 24 hours. To remove the tissues, the sampleswere centrifuged at 440 G at 4° C. for 10 minutes and the supernatantwas collected as skin extract. Ten pi of the skin extract obtained wasused as a sample and subjected to SDS-PAGE electrophoresis. The proteinsseparated within the gel were transferred onto a PVDF membrane using ablotting device (ATTO). The membrane was incubated with PBS containing3% skim milk and 0.1% Tween20 (S-T-PBS) at room temperature for onehour, and then was allowed to react with each of rabbit anti-mouse HMGB1antibody, rabbit anti-mouse HMGB2 antibody, or rabbit anti-mouse HMGB3antibody which were diluted 1,000-fold with S-T-PBS, at 4° C. for 16hours. After the reaction, the PVDF membrane was washed with S-T-PBSfive times for 5 minutes. Then, the PVDF membrane was incubated with2,000-fold diluted (diluted with S-T-PBS) peroxidase-labeled goatanti-rabbit IgG antibody (GE Healthcare) at 25° C. for 1 hour. Further,after washing with S-T-PBS five times for 5 minute, the PVDF membranewas allowed to react with ECL Western Blotting Detection System (GEHealthcare). The ECL film was exposed and developed to detect thepresence of HMGB1, HMGB2, and HMGB3 proteins.

RNA was extracted from the skin of neonatal mouse using Trizol(Invitrogen), and further cDNA was synthesized using the SuperScript IIIcDNA Synthesis Kit (Invitrogen). Using this cDNA as a template, cDNAs ofHMGB1, HMGB2, and HMGB3 were amplified using the polymerase chainreaction (PCR) method. The cDNAs were inserted into the plasmid vectorpCAGGS for expressing proteins in mammalian cells, such that proteinswith an additional Flag-tag sequence (Asp-Tyr-Lys-Asp-Asp-Asp-Lys; SEQID: 30) at the N-terminus of the amino acid sequence could be expressed.These plasmid vectors were transfected into HEK293 (Human embryonickidney derived culture cell line) and cultured for 48 hours to expressthe proteins. Cells expressing each of the HMGB1, HMGB2, and HMGB3proteins and the culture supernatant were incubated at 4° C. for 16hours, which was then centrifuged at 4,400 g for 5 minutes to collectthe supernatant. 100 μL of the anti-Flag antibody Gel (Sigma) was mixedinto 50 mL of this supernatant, and was then incubated at 4° C. for 16hours. Centrifugation was then performed to collect the gel, and washedwith PBS five times. Further, the protein was eluted using 3× Flagpeptide (final 100 μg/ml). Expressions of recombinant proteins wereobserved by the Western blot method using 1,000-fold diluted (dilutedwith S-T-PBS) mouse anti-Flag antibody and 2,000-fold diluted (dilutedwith S-T-PBS) peroxidase-labeled anti-mouse IgG antibody (GEHealthcare). The mouse bone marrow mesenchymal stem cell migrationactivity in these purified recombinant proteins was assessed using aBoyden chamber. Moreover, in order to observe the in vivo drug efficacyof the HMGB family, the dorsal skin of 8-week-old C57BL/6 mice was cutout in a circle having a diameter of 8 μm to prepare cutaneous ulcermodels. Purified HMGB1, HMGB2, and HMGB3 (100 ng/μl) were each mixedwith the same amount of hyaluronic acid solution having a concentrationof 1 g/100 mL of PBS, and 100 μL of it was administered to the ulcersurface. The ulcer surface was covered with a transparent adhesive wounddressing/protective material Tegaderm (3M Healthcare) to avoid drying,and the wound area was measured over time to determine the therapeuticeffect.

Further, to examine whether or not the human skin extract and thepurified human HMGB1 has an activity to allow migration of human bonemarrow mesenchymal stem cells, a Boyden chamber was used for assessment.A human skin having an area of 1 cm² was immersed in 1 ml PBS, and thenwas incubated at 4° C. for 16 hours and subsequently centrifuged at 440G at 4° C. for 10 minutes. The supernatant alone was collected to beused as a human skin extract. Moreover, human bone marrow mesenchymalstem cells (Cambrex) were used as the cells to be placed in the upperchamber of the Boyden chamber (as a result of surface antigen analysisby flow cytometry, these cells have been confirmed to be CD105-positive,CD166-positive, CD29-positive, CD44-positive, CD34-negative, andCD45-negative. They have also been found to differentiate intoadipocytes, chondrocytes, and osteocytes by differentiation inductiontests). Moreover, 100 ng/well of human HMGB1 (R&D) and human skinextract diluted 10-fold with PBS were placed in the lower chamber. PBSwas used as a control.

Results: As a result of Western blotting, bands of HMGB2 and HMGB3 weredetected as well as the HMGB1 band. Therefore, the neonatal mouse skinextract was confirmed to contain the family proteins, HMGB2 and HMGB3,besides HMGB1 (FIG. 8). Expression vectors of HMGB1/HMGB2/HMGB3 having aFlag-tag added at the N-terminus of each protein were prepared (FIG. 9).These expression vectors were transfected into HEK293 cells, and theexpressed proteins were purified using the Flag-tag, and Westernblotting was carried out to observe these proteins (FIG. 10). The mousebone marrow mesenchymal stem cell migration activity was measured usingthese purified proteins, and the activity was confirmed in all of theproteins (FIGS. 11A-11C). The ulcer area produced in the back of themouse was measured every 7 days, and a significant effect on reducingulcer area was confirmed in the HMGB1, 2, and 3 treatment groups, ascompared to the untreated group (FIGS. 12A-12C). Similar to the mousecase, human HMGB1 and the human skin extract were revealed to have theactivity of inducing the migration of human bone marrow mesenchymal stemcell (FIG. 13).

Discussion: HMGB2 and HMGB3 are known as proteins having high homologiesto HMGB1. These proteins are also expected to have properties similar toHMGB1. It was confirmed that HMGB2 and HMGB3 of the HMGB1 family arealso produced from the extract of the free skin section. Further,HMGB1/HMGB2/HMGB3 recombinant proteins were produced, and their in vitrobone marrow mesenchymal stem cell migration activity and the in vivotherapeutic effect on a cutaneous ulcer were also confirmed. It wasrevealed that the HMGB family (HMGB1/HMGB2/HMGB3) and the recombinantHMGB family in the neonatal mouse free skin section have a bone marrowmesenchymal stem cell-inducing activity and an activity of locallyinducing bone marrow-derived stem cells which are differentiatable intoepithelium, and that the thus induced bone marrow-derived cell groupdifferentiates into various cells such as epidermal keratinocytes, hairfollicles, and fibroblasts in the damaged tissue to promote the recoveryof the damaged tissue. Moreover, since bone marrow mesenchymal stemcells are multipotent stem cells, the present inventors believe thattherapeutic effects can also be expected in the same manner bysystematic administration or local administration of the HMGB family totreat damaged states in other tissues, for example, tissue damages suchas brain injury, myocardial infarction, and bone fracture.

Moreover, it is known that, between human and mouse, amino acid sequencehomology for HMGB1 is 98% (213/215), 96% (202/210) for HMGB2, and 97%(195/200) for HMGB3. Therefore, human HMGB and mouse HMGB are consideredto have similar activities, and the results revealed that human skinextract and human HMGB1 have bone marrow mesenchymal stem cell-inducingactivities in a manner same as those of mouse skin extract and mouseHMGB1.

Reference Example 2

Objective: Establishment of a method of producing a tissue extractcontaining bone marrow mesenchymal stem cell-inducing factors

Methods: Brain, heart, intestine, kidney, and liver of a 6-week-oldC57BL6 mouse and skin of a neonatal mouse were immersed in 1 ml ofphysiological phosphate buffer solution (PBS) at pH 7.4. The solutionswere incubated at 4° C. for 24 hours, and then centrifuged at 440 G at4° C. for 10 minutes to remove the tissues. The supernatants werecollected to prepare tissue extracts. To confirm whether the thusobtained extract has a bone marrow-derived mesenchymal stemcell-inducing activity, the migration activity of bone marrow-derivedmesenchymal stem cells was examined using a Boyden chamber. Moreover,the HMGB1 concentration contained in these samples was measured using anHMGB1 ELISA Kit (Shino-Test). Further, tissue extracts of the brain,heart, and skin were allowed to bind to a heparin affinity column, andthe bone marrow-derived mesenchymal stem cell-inducing activity in theprotein-bound fraction was confirmed using Boyden chamber.

Results: The mouse brain extract contained an amount of HMGB1 equivalentto the neonatal mouse skin extract. Further, bone marrow mesenchymalstem cell-inducing activity was also observed in the mouse brain as wellas in the skin. Although the mouse intestine extract and the mouse heartextract contained little HMGB1, bone marrow mesenchymal stemcell-inducing activities were observed. Moreover, the heparincolumn-bound fractions of mouse brain and mouse heart, as well as theheparin column-bound fraction of mouse skin, showed bone marrowmesenchymal stem cell-inducing activities (FIG. 14). Table 1 shows themeasurement results of the HMGB1 concentration and the bone marrowmesenchymal stem cell-inducing activity in each of the mouse tissueextracts.

TABLE 1 HMGB1 concentration Bone marrow-mesenchymal (ng/mL) stemcell-inducing activity Skin 110 Present Brain 140 Present Heart 4Present Intestine 0 Present Kidney 115 ND Liver 61 ND ND: Not Determined

DISCUSSION

A method in which HMGB1 can be conveniently extracted not only from theskin but also from the brain was developed by simply immersing theseorgans in a physiological buffer. This method is also applicable toother organs such as liver and kidney. Moreover, although the extractsfrom intestine and heart contain little HMGB1, a bone marrow mesenchymalstem cell-inducing activity was observed. This suggests these extractscontain other bone marrow mesenchymal stem cell-inducing substance(s)apart from HMGB1. Such substances contained in these extracts areoriginally present in each tissue, and are considered to physiologicallyinduce bone marrow mesenchymal stem cells to the damaged tissue when thetissue is damaged. The present invention developed a novel method forconveniently and functionally extracting from various organs multiplebone marrow mesenchymal stem cell-inducing substances including HMGB1.Further, a method for purifying bone marrow mesenchymal stemcell-inducing substances from a tissue extract using the binding to theheparin column was also developed. These substances having bone marrowmesenchymal stem cell-inducing activities can be purified from the brainand heart in the same manner as in the skin using a heparin column.

Reference Example 3

Objective: Establishment of a method for extracting mesenchymal stemcell migration activators from cultured cells.

Methods: Human embryonic kidney derived cultured cell line HEK293 andhuman cervix carcinoma cell line HeLa were each cultured in 10% fetalbovine serum-containing D-MEM (Nacalai). These cells were each washedwith PBS, and then 10⁷ cells were immersed in 5 ml of PBS (Nacalai) at4° C. for 16 hours. The solution was centrifuged at 440 G (accelerationof gravity) at 4° C. for 5 minutes, and then the supernatant wascollected. Human bone marrow mesenchymal stem cells were placed in theupper chamber of a Boyden chamber, and a 5-fold diluted (with DMEM) cellextract was placed in the lower chamber, to confirm the migrationactivity of human bone marrow mesenchymal stem cells.

Results: HEK293 extract and HeLa extract both showed similar bone marrowmesenchymal stem cell migration activities (FIGS. 15A-15B).

Discussion: Bone marrow mesenchymal stem cell migration activators weresuccessfully extracted by the convenient method of immersing culturedcells in PBS.

Reference Example 4

Objective: Whether or not regeneration of neural cells can be induced isexamined by producing mouse brain-defective models, to which aheparin-column purified fraction of skin extract is administered in asustained-release manner at the local lesion site, by which stem cellscontained in a mouse myeloid system is allowed to migrate into the locallesion site.

Methods:

(1) Preparation of Heparin-Column Purified Fraction of Skin Extract

An excised skin section of a neonatal mouse was incubated in PBS(mouse/ml) at 4° C. for 16 hours, and a skin extract was obtained. Theskin extract was diluted 10-fold with 9 volumes of 20 mM phosphatebuffer at pH 7.5 at 4° C. 20 mM phosphate buffer at pH 7.5 (30 ml) waspoured into HiTrap Hepalin HP column (column volume: 5 ml, GEHealthcare) in advance to equilibrate the column. The diluted solutionwas then allowed to bind to the column. Thereafter, the column waswashed with 20 mM phosphate buffer at pH 7.5 and 100 mM NaCl (30 ml). Toelute the adsorbed proteins, 20 mM phosphate buffer at pH 7.5 and 1,000mM NaCl were poured into the column, and the factions were eluted intothe tubes. Each of the adsorbed factions were evaluated according to themouse bone marrow-derived cell migration activity assessment using theBoyden chamber method, and fraction(s) having migratory activity wascollected. Solution(s) having the activity was used as a heparinpurified fraction(s) of the skin extract in the following ReferenceExample.

(2) Production of Myelosuppressive Mice

Mice were irradiated with single-dose of X-ray at 10 Gy to producemyelosuppressive mice.

(3) Transplantation of GFP Mouse Bone Marrow to Myelosuppressive Mice

Bone marrow cells were collected from both femurs and crus bones of GFPmice. These cells were administered to the myelosuppressive mice throughthe caudal vein 24 hours after the irradiation. The administration wascarried out under inhalational anesthesia using isoflurane.

(4) Production of a Brain-Defective (Brain Tissue-Defective) Mouse Model

The myelosuppressive mice transplanted with GFP mouse bone marrow cellswere subjected to inhalational anesthesia using isoflurane, andpentobarbital (45 mg/kg) was intraperitoneally injected to the mice. Themice were fixed onto a brain stereotaxis apparatus and subjected to amidline incision in the head with a scalpel. Trepanation was carried outat 2.5 mm right-lateral and 12.5 mm anterior to the bregma using a drill(FIG. 16A). At a 3 mm depth from this site, a 20G Surflow needle wasinserted and fixed. Then, a negative pressure was applied using asyringe to suck part of the brain tissue (FIG. 16B).

(5) Administration of a Heparin-Column Purified Fraction of Skin Extractto the Brain Tissue-Defective Site

Five microliters of a heparin-column purified fraction of skin extractdissolved in a fibrin tissue adhesive formulation (fibrinogen) (Bolheal(Kaketsuken)) was injected to the above site, and subsequently, 5 μl ofa fibrin tissue adhesive formulation (thrombin) (Bolheal (Kaketsuken))was injected using a Hamilton syringe and a 26G syringe (FIG. 16C). Theaim of this operation was to exert the sustained-release agent effect ofa heparin-column-purified fraction of the skin extract.

(6) Assessment of the Effects of Neural Cell Regeneration in BrainTissue-Defective Sites

Mice of the control group and the treatment group were used for theassessment. An appropriate elapsed time setting (over time) wasdetermined, the mice were perfused with 4% paraformaldehyde and fixedand then the brain was cut out. Further, external fixation was performedwith 4% paraformaldehyde. These were then dehydrated in a 15% and 30%sucrose gradient to produce frozen sections.

The nucleus were stained with a DAPI (4′,6-Diamidino-2-phenylindole,dihydrochloride) solution and the section was sealed using aphotobleaching inhibitor. The accumulation of GFP-positive cells in thelesion site (brain tissue-defective site) was assessed using a confocallaser microscope.

Results: The accumulation of GFP-positive cells is qualitatively shownfor 2 weeks, and 6 weeks after the administration. The accumulation ofGFP-positive cells tends to be higher in the lesion sites of thetreatment group rather than the control group, for both 2 weeks(control; FIG. 16D, skin extract heparin-column-purified fraction; FIG.16E) and 6 weeks (control; FIG. 16F, skin extractheparin-column-purified fraction; FIG. 16G) after the administration.

Discussion: The administration of the heparin-column-purified fractionof the skin extract resulted in the accumulation of bone marrow-derivedcells in the brain tissue-defective site, which showed a nerve cellform. Bone marrow-derived mesenchymal stem cells are also known todifferentiate into nerve cells and the result revealed that theheparin-column purified fraction of the skin extract is capable ofinducing neural cell regeneration of the injured site in the brain.Moreover, this is also applicable to neuronal regeneration of damagedsites in brain tissues in cerebral ischemic diseases and cerebralcontusions.

Reference Example 5

Purpose: To identify bone marrow-derived tissue stem cell-inducingfactors in skin tissue extracts

Methods: By the method described below, study was conducted to identifyfactors responsible for mobilizing bone marrow mesenchymal stem cells,which were predicted to be released from excised skin under hemostaticcondition.

(1) Bone marrow cells were harvested from the thighbones or crural bonesof C57BL/6 mice to obtain mouse bone marrow-derived mesenchymal stemcells. The cells were seeded into a cell culture dish with D-MEM(Nacalai) supplemented with 10% fetal bovine serum as a culture mediumand cultured at 37° C. under 5% carbon dioxide gas. When the cells weregrown to occupy an area of 70 to 100% relative to the bottom of theculture dish, the cells were detached from the culture dish using 0.25%trypsin/1 mM EDTA (Nacalai). The cells were then passaged under the sameculture conditions. After at least five passages, the adherent cellswere isolated and further cultured, and analyzed for cell surfaceantigens by flow cytometry. The result showed that the cells werepositive for CD44 and Sca-1, and negative for Lin, CD45, and c-kit. Itwas confirmed that the cells can differentiate into osteocytes andadipocytes and thus have the characteristics of bone marrow mesenchymalstem cells.

(2) Free skin pieces isolated from five heads of neonatal mice(two-day-old) were immersed in 5 ml of physiological phosphate bufferedsaline (PBS, pH 7.4). After 24 hours of incubation at 4° C., the samplewas centrifuged at 440 G at 4° C. for ten minutes to remove tissues. Thesupernatant was collected as skin extract. In addition, in the same way,free skin pieces isolated from a six-week-old mouse were immersed in 5ml of physiological phosphate buffered saline (PBS, pH 7.4). Afterincubation at 4° C. for 24 hours, the samples were centrifuged at 440 Gat 4° C. for ten minutes to remove tissues. The supernatants werecollected as skin extract.

(3) To confirm whether the prepared skin extract has the activity ofinducing bone marrow mesenchymal stem cells, the present inventors usedthe Boyden chamber to examine the chemotactic activity for previouslycloned bone marrow-derived mesenchymal cells derived from C57BL6 mice.Specifically, a mixture of DMEM (20 μl) and skin extract (5 μl) fromtwo-day-old or six-week-old mice was added into the bottom compartment(a volume of 25 μl) of a Boyden chamber, and a polycarbonate membranewith 8-μm micropores was placed on top. Then, the upper compartment (avolume of 50 μl) of the Boyden chamber was placed in contact with themembrane, and a suspension of bone marrow-derived mesenchymal stem cells(5×10⁴ cells/50 ml of culture medium (DMEM supplemented with 10% fetalbovine serum)) was added to the upper compartment. The chamber wasincubated in a CO₂ incubator at 37° C. for four to 24 hours. Afterincubation, the upper unit of the chamber was removed. The thin siliconefilm was detached and the number of bone marrow-derived mesenchymal stemcells migrating into the bottom compartment through the micropores wasquantitatively determined by staining the cells (FIG. 17).

(4) About 2-cm² skin specimens were excised from two-day-old andsix-week-old mice and immediately frozen in liquid nitrogen. The skinspecimens were crushed in a mortar. RNAs were extracted and purifiedfrom the samples using RNeasy (Qiagen). Using the purified RNAs,microarray assay was carried out to screen for mRNA expressed at higherlevels in the two-day-old mice. 767 genes showed two or more timesgreater scores in the two-day-old mice. Of these genes, proteins withhigh affinity for heparin, potential secretory proteins, and genes whosescores were six or more times greater in the two-day-old mice wereexamined and S100A9 was found as the 57^(th) gene from the top. Thus,S100A8 which is known to form a heterodimer with S100A9 in the skinextract from the two-day-old mice was detected by Western blotting.Specifically, 5 μl of the skin extract from the two-day-old mice wascombined with 5 μl of SDS-PAGE sample buffer (Bio-Rad). The mixture washeated in a heat block at 98° C. for five minutes, and then cooled to25° C. The resulting sample was applied onto 12.5% acrylamide gele-PAGEL (ATTO) and electrophoresed at 40 mA for 75 minutes using anelectrophoretic device (ATTO). The gel was collected afterelectrophoresis. Using a blotting device (ATTO), proteins in the gelwere transferred to PVDF membrane (7 cm by 9 cm, Millipore) pretreatedwith 100% methanol. After 75 minutes of protein transfer at 120 mA, thePVDF membrane was removed and shaken at room temperature for 30 minutesin PBS (Nacalai) containing 4% skim milk. Then, the removed PVDFmembrane was soaked in 5 μl of anti-S100A8 antibody (R&D) or anti-S100A9antibody (R&D) each diluted with 10 ml of PBS containing 4% skim milk,and shaken at room temperature for 60 minutes. After the antibodysolution was removed, the membrane was shaken in 30 ml of PBS containing0.1% Tween20 at room temperature for five minutes. This washing wasrepeated five times. Then, the membrane was soaked in 5 μl ofHRP-labeled anti-goat IgG antibody (GE healthcare) diluted with 10 ml ofPBS containing 4% skim milk, and shaken at room temperature for 45minutes. After the antibody solution was removed, the membrane waswashed with 30 ml of PBS containing 0.1% Tween20 at room temperature forfive minutes while shaking. This washing was repeated five times. Themembrane was treated for luminescence using ECL Detection Kit (GEhealthcare), and then exposed on a film. Signals for S100A8 and S100A9proteins were gained by developing the film in a developing apparatus(FIGS. 18A-18B).

(5) Factors having the activity of mobilizing bone marrow-derivedmesenchymal stem cells in skin extracts were purified by heparinaffinity column chromatography. The experiment described below wascarried out using an FPLC device (GE healthcare). First, the skinextract of two-day-old mice was diluted 10-fold with nine volumes of 20mM phosphate buffer (pH 7.5) at 4° C. (dilution solution A). 300 ml of20 mM phosphate buffer (pH 7.5) was run through a HiPrep 16/10 HeparinFF (GE Healthcare) column to equilibrate the column in advance, anddilution solution A was loaded onto the column. Then, the column waswashed with 300 ml of 20 mM phosphate buffer (pH 7.5). 20 mM phosphatebuffer (pH 7.5) containing 10 mM NaCl (solution A) and 20 mM phosphatebuffer (pH 7.5) containing 500 mM NaCl (solution B) were prepared toelute the adsorbed protein. Elution was started with [100% solution A+0%solution B], and then the proportion of solution B was graduallyincreased. Finally, the column was eluted with [0% solution A+100%solution B]. The total elution volume was 150 ml. The eluate wasfractionated into silicone-coated tubes (3 ml/tube). 5 μl each of thefractionated samples were mixed with 5 μl of SDS-PAGE sample buffer(Bio-Rad). The mixtures were heated in a heat block at 98° C. for fiveminutes, and then cooled to 25° C. The samples were applied onto anacrylamide gel e-PAGEL (5-20% gradient, ATTO), and electrophoresed at 40mA for 75 minutes using an electrophoresis device. After theelectrophoresis, the electrophoresed protein was detected using theDodeca Silver Stain Kit (Bio-Rad) (FIG. 19).

The chemotactic activity of fractionated samples was assayed in the sameway as described above using a Boyden chamber (FIG. 20).

The presence of S100A8 and S100A9 proteins in the fractionated sampleswas detected in the same way as described above by Western blotting(FIGS. 21A-21B).

(6) RNA was extracted from neonatal mouse skin using Trizol(Invitrogen), and then cDNA was synthesized from the RNA using theSuperScript 111 cDNA Synthesis Kit (Invitrogen). cDNAs of S100A8 andS100A9 were amplified by the polymerase chain reaction (PCR) methodusing the cDNA as a template. These cDNAs were each inserted into amammalian cell protein-expression plasmid vector, pCAGGS, to express theproteins in which a GST-tag sequence (amino acid sequence/SEQ ID NO: 31;DNA sequence/SEQ ID NO: 32) is attached to the N-terminus of their aminoacid sequences (FIG. 22). pCAGGS-GST-S100A8 or pCAGGS-GST-S100A9 wereeach transfected into a human fetal kidney cell-derived cultured cellline HEK293 using a lipofection reagent (Invitrogen). 48 hours aftertransfection, the cells and culture supernatant were collected, andcentrifuged at 4,400 G at 4° C. for five minutes. The supernatant(Supernatant A) and cells were collected separately. PBS containing 0.1%Tween20 was added to the cells, and the suspension was sonicated on icefor 30 seconds to disrupt the cell membrane. After centrifugation at4,400×g at 4° C. for five minutes, the resulting supernatant wascollected (Supernatant B). Supernatants A and B were combined togetherand loaded onto a HiTrap GST FF column (5 ml; GE Healthcare) whosebuffer had been replaced with 30 ml of PBS in advance. After loading,the column was washed with 100 ml of PBS, and the adsorbed protein waseluted with 20 mM phosphate buffer (pH 8) containing reducedglutathione. The chemotactic activity of recombinant S100A8 and S100A9for bone marrow mesenchymal stem cells was assessed using the Boydenchamber. The samples were prepared by dissolving purified S100A8 orS100A9 protein at 0.1 ng/μl in DMEM, or by diluting the skin extract oftwo-day-old mice with four volumes of DMEM, and added into the bottomcompartment of the Boyden chamber. A negative control prepared asfollows was used the same way: protein was extracted from cellstransfected with a control vector which does not carry the cDNA ofS100A8 or S100A9 as an insert; and then a fraction was eluted from aHiTrap GST FF column. After a sample was added into the bottomcompartment, a polycarbonate membrane with 8-μm micropores was placed ontop. Then, the upper unit (a volume of 50 μl) of Boyden chamber wasplaced in contact with the membrane, and a suspension of bonemarrow-derived mesenchymal stem cells (5×10⁴ cells/50 ml of culturemedium (DMEM supplemented with 10% fetal bovine serum)) was added to theupper chamber. The chamber was incubated in a CO₂ incubator at 37° C.for four to 24 hours. After incubation, the upper unit of the chamberwas removed. The thin silicone film was detached and the number of bonemarrow-derived mesenchymal stem cells migrating into the bottomcompartment through the micropores was quantitatively determined bystaining the cells (FIG. 23).

(7) Eight-week-old male mice were injected with 250 μl of theabove-described purified GST-S100A8 or S100A9 recombinant proteins (1ng/μl) via the caudal vein. 12 hours after injection 1 ml of peripheralblood was collected from the hearts of the mice under inhalationanesthesia with isoflurane using a 1-ml heparin-coated syringe. Theblood samples were each combined with 3 ml of PBS, and then gentlyoverlaid onto 3 ml of Ficoll (GE healthcare). The resulting samples werecentrifuged using centrifuge at 400×g at 25° C. for 40 minutes. Thecells in the opaque middle layer were collected as a mononuclear cellfraction. 1 ml of HLB solution (Immuno-Biological Laboratories Co.,Ltd.), a hemolytic agent, was added to the collected cells, and thecells were incubated at room temperature for five minutes. Thishemolytic treatment was repeated twice. After adding 10 ml of PBS, thecells were centrifuged at 440×g at 25° C. for five minutes. Theresulting supernatants were removed, and the cells were collected.1,000,000 cells were incubated at room temperature for 20 minutes with aPE-labeled anti-mouse PDGFRα antibody (e-Bioscience), PE-labeledanti-mouse PDGFRβ antibody (e-Bioscience), FITC-labeled anti-mouse CD45antibody (BD biosciences), and PerCy5-labeled anti-mouse CD44 antibody(BD biosciences), each diluted 100-fold with PBS. Then, the cells werecentrifuged at 440×g at 25° C. for five minutes. The supernatants wereremoved. 400 μl of PBS containing 1% paraformaldehyde was added to thecells to prepare samples for flow cytometric analysis. Antibodies wereused in the following combinations:

(I) PDGFRα/CD45/CD44

(II) PDGFβ/CD45/CD44

The ratio of cells expressing PDGFRα (or β) and CD44 to cells that wereweakly positive or negative for CD45 was determined based on theanalysis result (FIGS. 24A and B).

Results: Skin samples excised from two-day-old and six-week-old micewere assessed for the activity of mobilizing bone marrow mesenchymalstem cells. The activity of skin extract from two-day-old mice wasdemonstrated to be stronger than that of the skin extract fromsix-week-old mouse. Strong S100A9 expression in the skin fromtwo-day-old mice was found by DNA microarray analysis. Crude samples ofskin extracts purified on a heparin column exhibited correlation betweenthe migrating activity of mesenchymal stem cells and the contents ofS100A9 and S100A8. Expression vectors for these proteins wereconstructed, and the recombinant proteins were produced using HEK293 andpurified. The migrating activity of bone marrow mesenchymal stem cellswas confirmed in the purified S100A8 and S100A9 samples by assays usingBoyden chamber. Furthermore, when intravenously administered to mice,the proteins also exhibited the activity of mobilizing a population ofPDGFRα and CD44 double-positive cells to peripheral blood (FIGS.24A-24B).

Discussion: The present inventors for the first time in the worlddiscovered in the present invention that free skin pieces produce S100A8and S100A9, and the produced S100A8 and S100A9 proteins had strongactivities of mobilizing bone marrow-derived mesenchymal stem cells.Meanwhile, bone marrow mesenchymal stem cells are known as pluripotentstem cells that differentiate into bone tissues, adipose tissues,cartilage tissues, fibroblasts, and the like. Recently, it has beenindicated that bone marrow-derived cells also include pluripotent stemcells that differentiate into tissues such as cardiac muscle, nervecells, and epidermal cells. Since the present invention demonstratesthat the epidermal cells, hair follicle cells, fibroblasts ofsubcutaneous tissues, and such in the grafted skin are constituted bybone marrow-derived cells, S100A8 and S100A9 can be speculated to beresponsible for mobilizing bone marrow-derived tissue stem cells to theskin graft to induce functional repair of damaged tissues. Even byintravenous injection, S100A8 and S100A9 can mobilize bone marrowmesenchymal stem cells to peripheral blood. Thus, S100A8 and S100A9 canalso be administered via peripheral circulation to tissues located deepinside the body where local administration is difficult (brain, heart,spinal cord, etc.). The present inventors believe that effects such asshortening the healing time, functional regeneration of damaged tissues,and such can be expected in the healing process for not only damagedskin tissues but also various damaged tissues such as brain, muscle, andbone by using the present invention in pharmaceuticals, which enableslocal mobilization of the bone marrow-derived tissue stem cellsincluding mesenchymal stem cells in regeneration of damaged tissues.

Reference Example 6

Purpose: To assess the therapeutic effect of S100A8 on cutaneous ulcerin normal and diabetic mice

Methods: Recombinant S100A8 protein was administered to cutaneous ulcermodel mice to assess its therapeutic effect on ulcer. Test mice usedwere: C57/B16 mice transplanted with bone marrow cells expressing GFP,and BKS.Cg-m+/+Leprdb/J (db mice), which are diabetes model mice.Cutaneous ulcers with a diameter of 6 mm were formed on the skin of themice. When cutaneous ulcer is formed in mice, the surrounding skin closeto the skin defect rapidly shrinks. In this experiment, to create atherapeutic model for skin defect, in which skin defect is treated notthrough shrinkage but by covering it with regenerated skin, a siliconerubber disc with an outer diameter of 10 mm, inner diameter of 6 mm, andthickness of 0.5 mm was fixed at the skin defect site to the skinsurrounding the ulcer using an adhesive agent for skin surgery (Aronalpha A) and nylon suture to prepare a model for treating skin defect bycovering it with regenerated skin, not by shrinkage of the skin. Then,the recombinant S100A8 protein was directly administered to the ulcersurface at 1.5 μg/day every day for seven days. Furthermore, the ulcersurface was protected with film dressing Tegaderm (3M) to preventdesiccation of the ulcer surface. The ulcer surface area was measuredover time to assess the therapeutic effect.

Results and discussion: In normal mice, the ulcer surface area wassignificantly reduced in the S100A8-treated group seven days after thestart of treatment as compared to the control group (FIG. 25).Furthermore, in diabetes mice, the ulcer surface area was alsosignificantly reduced in the S100A8-treated group seven days after thestart of treatment as compared to the control group (FIG. 26). In otherwords, the significant cutaneous ulcer-reducing effect was observed inboth normal and diabetes mice. From this result, it is confirmed thatS100A8 has the therapeutic effect on cutaneous ulcer in not only normalmice but also diabetes mice.

Reference Example 7

Purpose: Mobilization of bone marrow tissue stem cells to peripheralblood using bone marrow-derived tissue stem cell-inducing factors inskin tissue extract

Methods: To achieve the above purpose, a study was conducted by themethod described below.

(1) Preparation of bone marrow-derived tissue stem cell-inducer. Freeskin pieces isolated from 25 neonatal mice (two days old) were immersedin 25 ml of phosphate buffered saline (PBS), pH 7.4. After 24 hours ofincubation at 4° C., the sample was centrifuged at 440 G at 4° C. forten minutes to remove the tissue. The supernatant was collected as skinextract (SE).

Meanwhile, RNA was extracted from neonatal C57/B16 mice skin usingTrizol (Invitrogen), and then cDNA was synthesized using the SuperScriptIII cDNA Synthesis Kit (Invitrogen). Polymerase chain reaction (PCR) wascarried out using this cDNA as a template to amplify HMGB1 cDNA. TheHMGB1 cDNA was inserted into an mammalian cell protein expressionplasmid vector, pCAGGS, to express a protein in which a Flag-tagsequence (Asp-Tyr-Lys-Asp-Asp-Asp-Lys, SEQ ID NO: 30) is attached to theN-terminus of its amino acid sequence (FIG. 32). The plasmid vector wastransfected into HEK293 (cultured cell line derived from human fetalkidney cell). The cells were cultured for 48 hours to express theprotein. Each sample of cells expressing the HMGB1 protein and theculture supernatant were incubated at 4° C. for 16 hours, and thencentrifuged at 4,400×g for five minutes. The supernatant was collected,and anti-Flag Antibody Gel (Sigma) was added thereto in an amount of 100μl per 50 ml of the supernatant. The mixture was incubated at 4° C. for16 hours. The gel was collected by centrifugation, followed by five PBSwashes. Then, the gel was eluted with 3× Flag peptide (final 100 μg/ml).The concentration of the eluted protein was determined using the HMGB1ELISA Kit (Shino-Test Co.). After freeze-drying, the proteinconcentration was adjusted to 200 μg/ml with PBS.

(2) Eight-week-old male mice (C57/B16) were administered with 500 μl ofthe above-described skin extract (SE), or 500 μl of PBS as a negativecontrol group, via the caudal vein using syringes attached with a 30G ½injection needle (FIG. 33). Six, 12, 24, and 48 hours afteradministration, 1 ml of peripheral blood was collected from the heartsof the mice under inhalation anesthesia with isoflurane using aheparin-coated 1-ml syringe. The blood samples were each combined with 3ml of PBS, and then gently overlaid onto 3 ml of Ficoll (GE healthcare).The resulting samples were centrifuged using a centrifuge at 400×g at25° C. for 40 minutes. The cells in the opaque middle layer werecollected as a mononuclear cell fraction. 1 ml of HLB solution(Immuno-Biological Laboratories Co., Ltd.), a hemolytic agent, was addedto the collected cells. The cells were incubated at room temperature forfive minutes. This hemolytic treatment was repeated twice. After adding10 ml of PBS, the cells were centrifuged at 440×g at 25° C. for fiveminutes. The supernatants were removed, and the cells were collected.1,000,000 cells were incubated at room temperature for 20 minutes withantibodies each diluted 100-fold with PBS including a PE-labeledanti-mouse PDGFRα antibody (e-Bioscience), PE-labeled anti-mouse PDGFRβantibody (e-Bioscience), and PerCy5-labeled anti-mouse CD44 antibody (BDbiosciences). After incubation, the cells were centrifuged at 440×g at25° C. for five minutes. The supernatant was removed. 400 μl of PBScontaining 1% paraformaldehyde was added to the cells to prepare asample for flow cytometric analysis.

Eight-week-old male mice (C57/B16) were administered with 250 μl ofmouse HMGB1 (1 μg/μl), or 250 μl of PBS as a negative control group, viathe caudal vein using syringes attached with a 30G ½ injection needle(FIG. 35). 12 hours after administration, 1 ml of peripheral blood wascollected from the hearts of the mice under inhalation anesthesia withisoflurane using a heparin-coated 1-ml syringe. The blood samples wereeach combined with 3 ml of PBS, and then gently overlaid onto 3 ml ofFicoll (GE healthcare). The resulting samples were centrifuged in acentrifuge at 400×g at 25° C. for 40 minutes. The cells in the opaquemiddle layer were collected as a mononuclear cell fraction. 1 ml of HLBsolution (Immuno-Biological Laboratories Co., Ltd.), a hemolytic agent,was added to the collected cells. The cells were incubated at roomtemperature for five minutes. This hemolytic treatment was repeatedtwice. After adding 10 ml of PBS, the cells were centrifuged at 440×g at25° C. for five minutes. The supernatants were removed, and the cellswere collected. 1,000,000 cells were incubated at room temperature for20 minutes with antibodies each diluted 100-fold with PBS including aPE-labeled anti-mouse PDGFRα antibody (e-Bioscience) and PerCy5-labeledanti-mouse CD44 antibody (BD biosciences). After incubation, the cellswere centrifuged at 440×g at 25° C. for five minutes. The supernatantwas removed. 400 μl of PBS containing 1% paraformaldehyde was added tothe cells to prepare a sample for flow cytometric analysis.

Results: PDGFRα and CD44 double-positive cells were demonstrated to besignificantly mobilized to peripheral blood 12 hours after injection ofthe skin extract (SE) (FIG. 34). Furthermore, PDGFRα and CD44double-positive cells were demonstrated to be significantly mobilized toperipheral blood 12 hours after injection of HMGB1 (FIG. 36).

Reference Example 8

Purpose: To test whether mesenchymal stem cells are mobilized toperipheral blood by intravenous administration of recombinant HMGB1protein.

Methods: C57BL6 mice (eight to ten weeks old, male) were administeredwith 400 μl of physiological saline containing 100 μg/ml recombinantHMGB1 protein (40 μs of HMGB1) or 400 μl of physiological saline alonethrough the caudal vein. After 12 hours, peripheral blood was collectedfrom the mice. The blood samples were diluted with PBS to a total volumeof 4 ml. The diluted blood samples were overlaid onto 3 ml ofFicoll-Paque Plus (GE) placed in centrifuge tubes. The samples werecentrifuged at 100 G at 18° C. for ten minutes. The middle layercontaining mononuclear cells was transferred to a fresh centrifuge tube,and 45 ml of PBS was added thereto. The tube was centrifuged at 440 G at18° C. for five minutes. The supernatant was removed. Again, 45 ml ofPBS was added, and the tube was centrifuged at 440 G at 18° C. for fiveminutes. The supernatant was removed. The prepared mononuclear cellswere incubated with Phycoerythrobilin (PE)-labeled anti-mouse PDGFRαantibody and Allophycocyanin (APC)-labeled anti-mouse CD44 antibody.Then, the abundance of PDGFRα and CD44 double-positive cells in themononuclear cell fraction was assessed by flow cytometry (Facscan;Becton, Dickinson and Company).

Results: PDGFRα and CD44 double-positive cells, and PDGFRα-positive,CD44-negative cells in the peripheral blood mononuclear cell fractionwere demonstrated to be significantly increased 12 hours after HMGB1administration (FIGS. 37A-37C). Specifically, HMGB1 was demonstrated tohave the activity of mobilizing PDGFRα-positive cells to peripheralblood from bone marrow. PDGFRα is known as a mesenchymal stem cellmarker.

Discussion: PDGFRα and CD44 are known as surface markers of bone marrowmesenchymal stem cells, which are representative of bone marrow-derivedpluripotent stem cells. Bone marrow mesenchymal stem cells arepluripotent stem cells capable of differentiating into nerve cells,epithelial cells, or such as well as osteocytes, chondrocytes, andadipocytes. Meanwhile, the skin pieces used in this experiment are in anischemic condition. Thus, the tissues gradually necrotize andintracellular proteins such as nuclear proteins as well as cell surfaceproteins are released to the outside. HMGB1 is a protein contained inthe skin extract. In skin grafting or the like, such proteins serve as asignal to mobilize bone marrow-derived tissue stem cells into graftedskin. It is thus speculated that functional skin regeneration isachieved in the skin graft due to reconstitution of epidermis,hypodermis, follicular tissues, or such stemmed from the bone marrowcells. Based on this experiment, the present invention for the firsttime successfully discovered that bone marrow-derived tissue stem cellsare mobilized into peripheral blood circulation by intravenousadministration of HMGB1 or skin extract as described above. Thisdiscovery enables new therapeutic methods for treating intractablediseases with tissue damages such as brain infarction, myocardialinfarction, bone fracture, and cutaneous ulcer, which are based onmobilization of bone marrow-derived pluripotent stem cells intoperipheral blood. In addition, cells mobilized to peripheral blood canbe collected in the same way as conventional method for bloodcollection. Thus, the present invention enables simpler and safermethods for collecting bone marrow-derived tissue stem cells as comparedto the conventional method for treating brain infarction in which cellsare collected from the bone marrow.

INDUSTRIAL APPLICABILITY

The present invention enables collection of biologically functionalcells in a minimally invasive manner, which was difficult previously.This can be expected to promote basic studies using biologicallyfunctional cells as well as regeneration medicine using the collectedcells. Thus, the present invention is expected to be an innovativetechnique that provides patients suffering from intractable diseaseswith a new type of remedy. Furthermore, the vessels to be implanted inthe body serve as a seed for the development of novel medical materials,and thus are expected to contribute to the industrial development inthis field. G6-A0802Psq.txt

The invention claimed is:
 1. A method for collecting bone-marrow-derivedstem cells from a subject, wherein the method comprises: (I) implantingfor at least 12 hours a vessel that contains a substance of any of (a)to (c) below, completely under the skin in subcutaneous adipose tissueof the subject to induce stem cells present in the bone marrow of thesubject to migrate into the vessel implanted in subcutaneous adiposetissue, wherein, as a result of the presence of the substance, stemcells present in bone marrow of the subject enter the vessel implantedin subcutaneous adipose tissue: (a) hyaluronic acid, (b) an extract of acell or tissue, and (c) a heparin-binding fraction of a cell or tissueextract, wherein the vessel is made from a material that is biologicallyhypoallergenic when implanted into the subject without any of (a) to(c), and wherein the vessel has no openings other than at one end, andthe vessel has one closed end to form an area therein to collect thebone-marrow-derived stem cells; and (II) collecting bone-marrow-derivedstem cells from the vessel.
 2. The method according to claim 1, whereinthe extract of a cell or tissue is an extract of a live skin tissue orblood.
 3. The method of claim 1, in which the extract of a cell ortissue is produced by a method comprising the step of immersing a cellor tissue in a solvent.
 4. The method of claim 1, in which theheparin-binding fraction of the extract of a cell or tissue is producedby a method comprising the steps of: (a) immersing a cell or tissue in asolvent; (b) contacting immobilized heparin with an extract prepared instep (a); and (c) eluting a heparin-binding fraction from theimmobilized heparin.
 5. The method of claim 1, wherein the methodcomprises a step of removing the vessel to outside the body from underthe skin in subcutaneous adipose tissue of the subject after step (I)and before step (II).
 6. The method of claim 1, wherein step (II) is astep of collecting bone-marrow-derived stem cells from the vessel, whichis still under the skin in subcutaneous adipose tissue of the subject.7. A method for collecting bone-marrow-derived stem cells from asubject, wherein the method comprises: (I) implanting for at least 12hours a portion of a vessel in which the portion contains a substance ofany of (a) to (c) below, under the skin in subcutaneous adipose tissueof the subject to induce stem cells present in the bone marrow of thesubject to migrate into the portion implanted in subcutaneous adiposetissue, wherein, as a result of the presence of the substance, stemcells present in bone marrow of the subject enter the portion implantedin subcutaneous adipose tissue: (a) hyaluronic acid, (b) an extract of acell or tissue, and (c) a heparin-binding fraction of a cell or tissueextract, wherein the vessel is made from a material that is biologicallyhypoallergenic when implanted into the subject without any of (a) to(c), and wherein the vessel has no openings other than at one end, andthe vessel has one closed end to form an area therein to collect thebone-marrow-derived stem cells; and (II) collecting bone-marrow-derivedstem cells from the vessel.
 8. The method according to claim 7, whereinthe extract of a cell or tissue is an extract of a live skin tissue orblood.
 9. The method of claim 7, in which the extract of a cell ortissue is produced by a method comprising the step of immersing a cellor tissue in a solvent.
 10. The method of claim 7, in which theheparin-binding fraction of the extract of a cell or tissue is producedby a method comprising the steps of: (a) immersing a cell or tissue in asolvent; (b) contacting immobilized heparin with an extract prepared instep (a); and (c) eluting a heparin-binding fraction from theimmobilized heparin.
 11. The method of claim 7, wherein the methodcomprises a step of removing the portion to outside the body from underthe skin in subcutaneous adipose tissue of the subject after step (I)and before step (II).
 12. The method of claim 7, wherein step (II) is astep of collecting bone-marrow-derived stem cells from the vessel, theportion of which is still under the skin in subcutaneous adipose tissueof the subject.
 13. A method for collecting bone-marrow-derived stemcells from a subject, wherein the method comprises: (I) implanting forat least 12 hours a vessel that contains a substance selected fromhyaluronic acid and a heparin-binding fraction of a cell or tissueextract, completely under the skin in subcutaneous adipose tissue of thesubject to induce stem cells present in the bone marrow of the subjectto migrate into the vessel implanted in subcutaneous adipose tissue,wherein, as a result of the presence of the substance, stem cellspresent in bone marrow of the subject enter the vessel implanted insubcutaneous adipose tissue, wherein the vessel is made from a materialthat is biologically hypoallergenic when implanted into the subjectwithout the substance, and wherein the vessel has one closed end to forman area therein to collect the bone-marrow-derived stem cells; and (II)collecting bone-marrow-derived stem cells from the vessel.
 14. A methodfor collecting bone-marrow-derived stem cells from a subject, whereinthe method comprises: (I) implanting for at least 12 hours a portion ofa vessel in which the portion contains a substance selected fromhyaluronic acid and a heparin-binding fraction of a cell or tissueextract, under the skin in subcutaneous adipose tissue of the subject toinduce stem cells present in the bone marrow of the subject to migrateinto the portion implanted in subcutaneous adipose tissue, wherein, as aresult of the presence of the substance, stem cells present in bonemarrow of the subject enter the portion implanted in subcutaneousadipose tissue, wherein the vessel is made from a material that isbiologically hypoallergenic when implanted into the subject without thesubstance, and wherein the vessel has one closed end to form an areatherein to collect the bone-marrow-derived stem cells; and (II)collecting bone-marrow-derived stem cells from the vessel.
 15. A methodfor collecting bone-marrow-derived stem cells from a subject, whereinthe method comprises: (I) implanting for at least 12 hours a vessel thatcontains a substance selected from hyaluronic acid and a heparin-bindingfraction of a cell or tissue extract, completely under the skin insubcutaneous adipose tissue of the subject to induce stem cells presentin the bone marrow of the subject to migrate into the vessel implantedin subcutaneous adipose tissue, wherein, as a result of the presence ofthe substance, stem cells present in bone marrow of the subject enterthe vessel implanted in subcutaneous adipose tissue, wherein the vesselis made from a material that is biologically hypoallergenic whenimplanted into the subject without the substance, and wherein the vesselhas no openings other than at one end, and the vessel has one closed endto form an area therein to collect the bone-marrow-derived stem cells;and (II) collecting bone-marrow-derived stem cells from the vessel. 16.A method for collecting bone-marrow-derived stem cells from a subject,wherein the method comprises: (I) implanting for at least 12 hours aportion of a vessel in which the portion contains a substance selectedfrom hyaluronic acid and a heparin-binding fraction of a cell or tissueextract, under the skin in subcutaneous adipose tissue of the subject toinduce stem cells present in the bone marrow of the subject to migrateinto the portion implanted in subcutaneous adipose tissue, wherein, as aresult of the presence of the substance, stem cells present in bonemarrow of the subject enter the portion implanted in subcutaneousadipose tissue, wherein the vessel is made from a material that isbiologically hypoallergenic when implanted into the subject without thesubstance, and wherein the vessel has no openings other than at one end,and the vessel has one closed end to form an area therein to collect thebone-marrow-derived stem cells; and (II) collecting bone-marrow-derivedstem cells from the vessel.
 17. A method for collectingbone-marrow-derived stem cells from a subject, wherein the methodcomprises: (I) implanting for at least 12 hours a vessel that containshyaluronic acid, completely under the skin in subcutaneous adiposetissue of the subject to induce stem cells present in the bone marrow ofthe subject to migrate into the vessel implanted in subcutaneous adiposetissue, wherein, as a result of the presence of hyaluronic acid, stemcells present in bone marrow of the subject enter the vessel implantedin subcutaneous adipose tissue, wherein the vessel is made from amaterial that is biologically hypoallergenic when implanted into thesubject without hyaluronic acid, and wherein the vessel has one closedend to form an area therein to collect the bone-marrow-derived stemcells; and (II) collecting bone-marrow-derived stem cells from thevessel.
 18. A method for collecting bone-marrow-derived stem cells froma subject, wherein the method comprises: (I) implanting for at least 12hours a portion of a vessel in which the portion contains hyaluronicacid, under the skin in subcutaneous adipose tissue of the subject toinduce stem cells present in the bone marrow of the subject to migrateinto the portion implanted in subcutaneous adipose tissue, wherein, as aresult of the presence of hyaluronic acid, stem cells present in bonemarrow of the subject enter the portion implanted in subcutaneousadipose tissue, wherein the vessel is made from a material that isbiologically hypoallergenic when implanted into the subject withouthyaluronic acid, and wherein the vessel has one closed end to form anarea therein to collect the bone-marrow-derived stem cells; and (II)collecting bone-marrow-derived stem cells from the vessel.
 19. A methodfor collecting bone-marrow-derived stem cells from a subject, whereinthe method comprises: (I) implanting for at least 12 hours a vessel thatcontains hyaluronic acid, completely under the skin in subcutaneousadipose tissue of the subject to induce stem cells present in the bonemarrow of the subject to migrate into the vessel implanted insubcutaneous adipose tissue, wherein, as a result of the presence ofhyaluronic acid, stem cells present in bone marrow of the subject enterthe vessel implanted in subcutaneous adipose tissue, wherein the vesselis made from a material that is biologically hypoallergenic whenimplanted into the subject without hyaluronic acid, and wherein thevessel has no openings other than at one end, and the vessel has oneclosed end to form an area therein to collect the bone-marrow-derivedstem cells; and (II) collecting bone-marrow-derived stem cells from thevessel.
 20. A method for collecting bone-marrow-derived stem cells froma subject, wherein the method comprises: (1) implanting for at least 12hours a portion of a vessel in which the portion contains hyaluronicacid, under the skin in subcutaneous adipose tissue of the subject toinduce stem cells present in the bone marrow of the subject to migrateinto the portion implanted in subcutaneous adipose tissue, wherein, as aresult of the presence of hyaluronic acid, stem cells present in bonemarrow of the subject enter the portion implanted in subcutaneousadipose tissue, wherein the vessel is made from a material that isbiologically hypoallergenic when implanted into the subject withouthyaluronic acid, and wherein the vessel has no openings other than atone end, and the vessel has one closed end to form an area therein tocollect the bone-marrow-derived stem cells; and (II) collectingbone-marrow-derived stem cells from the vessel.